Method for recombinant microorganism expression and isolation of collagen-like polypeptides

ABSTRACT

A composition comprising a purified collagen-like polypeptide suitable as a peptizer, said polypeptide comprising [Gly-X-Y]n repeats, wherein Gly stands for glycine, X and Y represent any amino acid and n is an integer and selected such that the length of the [Gly-X-Y]n repeat is at least 2.5 kDa and wherein the amino acid sequence of said [Gly-X-Y]n repeats comprises more than 4 different amino acids and wherein said purified polypeptide is free of helix structure.

The present application is a divisional of U.S. application Ser. No.10/342,331, filed Jan. 15, 2003 (pending), which is a continuation ofU.S. application Ser. No. 09/617,842, filed Jul. 17, 2000 (abandoned),which is a divisional of U.S. application Ser. No. 09/219,849, filedDec. 23, 1998 (now U.S. Pat. No. 6,150,081), which claims benefit of NL1007908, filed Dec. 24, 1997, the entire contents of each of which ishereby incorporated by reference in this application.

SUMMARY OF THE INVENTION

The subject invention is directed at improved photographic products andimproved methods of production of said products. In particular theimprovement is arrived at through use of recombinant DNA technology inproduction of a component of a photographic product. The component ofinterest is collagen.

BACKGROUND OF THE INVENTION

The process of photographic product making is a complex procedure aboutwhich a lot has been disclosed and patented. In general terms theprocess to manufacture a photographic product like a photographic paperor triacetate cellulose film consists of coating several layers on topof either a laminated paper or a transparent polymer support. Theselayers are known as emulsion layers which can contain the radiationsensitive silver halide crystals as the most essential component orintermediate layers without these photosensitive components. The subjectinvention is directed at improving the photosensitive layer as such andimproving the production process of photographic layers.

There are several stages at which gelatin is used in the process of filmmaking. The function of the gelatin is different in each stage and thusthe required characteristics for each stage are different and it is tobe expected that collagen like substances can be specifically tailoredto suit each particular application.

A lot of attention has been focussed on the process of making silverhalide emulsions for photographic applications. A lot of attention hasbeen paid to the role of grain morphology of silver halide crystals andaspects that influence the AgX nucleation process and the subsequentripening process. The most essential component in an emulsion layer of aphotographic product consists of radiation sensitive silver bromide,silver chloride or silver bromochloride microcrystals optionallycontaining iodide which are commonly referred to as silver halidegrains. A peptizer is introduced during the precipitation of the grainsto avoid uncontrolled coalescence which coalescence will otherwiseexhibit a number of disadvantages i.a. limit the formation of thinintermediate and high aspect ratio tabular grain emulsions which in turnis disadvantageous in photography. Gelatin in numerous forms has beenused in photographic manufacturing processes as peptizer. It is wellknown that the tabular grains with high aspect ratio have severalphotographic advantages like increased sharpness, improved spendgranularity relationships, increased blue and minus blue speedseparation, more rapid developability and higher silver covering power(Research Disclosure Vol. 225 Jan. 1983, Item 22534; EP-A-0.610.796). Ithas also been desired to produce tabular grains not only with highaspect ratio but also with a narrow grain size distribution otherwiseexpressed as a desire for mono or homodispersity.

To date the gelatin used in commercial processes has been derived fromanimal sources in general simply by derival from animal bone and hide.The disadvantages of this material are the presence of impurities andthe fact that the nature of the composition is unclearly defined andthus not reproducible. It is unclear what components are present and inwhich amounts. In addition it is also unclear which components actuallyare required for optimal activity. The reproducibility of thephotographic manufacturing process is questionable due to the lack ofconsistency of the gelatin composition used at various stages of thephotographic manufacturing process.

The disadvantages of gelatin in photographic applications have beenaddressed in detail over the years and have been the subject of variouspatent applications. Most of these documents have been directed ataddressing processes of developing modified gelatins after the derivalthereof from the animal source to introduce particular improvements incharacteristics of the modified gelatins. In 1984 U.S. Pat. No.4,439,520 describes a desirability for more than 50% a of the crystalsto have aspect ratios higher than 8 as this would increase blue speed.In 1987 U.S. Pat. No. 4,713,320 mentions using gelatin with a methioninecontent below 30 micromoles per gram, preferably less than 5 micromolesper gram to arrive at thin trapezoidal, hexagonal and triangular tabulargrains. A normal bone delved gelatin was used which had been oxidized inorder to achieve a level of methionine below 30 micromoles per gram ofgelatin. The lower methionine content is also described in U.S. Pat. No.4,914,014 in 1990 as offering a wider range of pBr during precipitationconditions. Numerous publications cover processes for reducing themethionine content of gelatin. EP-558.410 published in 1993 describesoxidizing reagent reaction of alkali hypochlorite or H₂O₂ as do articlesin J. Photo. Sci 40 230-230 (Nippi), J. Photo. Sci 37, 14-18 (AGFA) of1992 and J. Imag. Sci 33,1 3-17 of 1989. Even as early as 1959 oxidationwas suggested as a manner to remove impurities.

There has also been a lot of research carried out on collagen andcollagen like proteins per se using recombinant nucleic acid technology.The use of recombinant DNA technology in combination with collagen andthe application thereof in photographic application has however beenremarkably absent. Most of the documents published in the area ofrecombinant collagen have been directed at diagnostic applications usingPCR technology on genomic nucleic acid not even requiring expression ofthe collagen encoding sequence. The mere presence of the sequence in thegenome suffices for diagnosis in these instances. Any such documentsactually mentioning expression of collagen encoding sequences certainlyhave not required a high degree or expression. Alternatively whereexpression is mentioned merely portions of the encoding sequence areexpressed rather than the complete sequence. Often these partialsequences are used for eliciting antibodies for which amounts ofproteinaceous material required are minimal. In addition once theantibodies are obtained the sequences are not required further forpharmaceutical application. Therefore in these instances low expressionis not a relevant issue. Also the expression of small portions of theencoding sequence can be expected to eliminate expression problems whichare attributed to the high degree of repetitivity of the encodingsequence.

Synthetic nucleic acid has been designed in an effort to overcomeexpression problems associated with longer repetitive sequences and alsoin an effort to design new types of protein i.e. synthetic protein. Suchsynthetic polypeptides are however extremely expensive to produce. It isthus not feasible to apply such in applications requiring large scaleproduction such as required in the field of photographic filmproduction.

Most applications of the prior art thus either do not require the highdegree of expression required for production on an industrial scale ordo not in fact provide the desired result. The documents discussingthese types of application have consequently either not addressed orhave not solved the problem of obtaining high expression of nativecollagen sequences or sequences of corresponding length and structure.

In general where the prior art has suggested expressing native sequencesor parts thereof general terms have been used and referrals to handbooksfor general transformation protocols have been given without furtherdetail. Any examples provided have used E. coli or S. cerevisiae asproducing organism and degree of expression has been of minor importanceor has not been focussed on.

Where expression problems of collagen like protein have been addressedthis has occurred by using either modified E. coli or higher animalcells and insect cells. The latter are also modified for posttranslational processing. The application of the latter type of cells ishowever prohibitive for large scale production due to the high costs ofthe cells, the media and the isolation of product. The disadvantage ofE. coli is that it cannot secrete the desired product. In addition therepetitivity of a nucleic acid sequence to be expressed will provideinstability of the transformed bacterium and thus result in lowexpression levels for any collagen like encoding sequence. It is thusnot feasible to apply such production (micro)organisms in applicationsrequiring large scale production as required in the field ofphotographic film production.

A lot of research effort has been directed at achieving the posttranscription modification required to arrive at fibrillar or triplehelix collagen which is the state of collagen as present in animalsources i.e. the state of collagen currently applied in industrial scalephotographic applications. It has been generally accepted that hostcells comprising post translational processing apparatus as suchthemselves or through addition of encoding sequences for posttranslational processing enzymes should be used when expressing collagenlike material in order to arrive at collagen with a triple helix andmore particularly to arrive at fibrillar collagen. It is commonlyaccepted that this form of collagen is the useful form for application.

Where the prior art has actually tentatively actually suggestedrecombinant collagen could be used in photographic applications therelevance of the particular form of collagen material has usually notbeen addressed vis a vis the requirements specific for such application.Some patent applications have mentioned in passing the use ofrecombinant collagen for photographic film, some even specificallymention photographic application. The teachings of such documents areclearly however directed at other issues and are not directedspecifically at photographic applications and the special requirementsthereof. Closer analysis actually shows that for various reasons none ofthe examples provided in such applications are in fact even suited forapplication in photographic films. The patent applications areconsidered non enabling and merely speculative of nature when it comesto applying recombinant collagen in photographic manufacturingprocesses. Examples of such documents are now provided.

WO90/05177 describes the production of novel polymers comprising smallrepeating sequences, specifically repeating groups such as silk aredisclosed. Collagen is suggested as one of the structures capable ofproviding a repeating unit. It is stated “The properties of CLP weredesigned so they would undergo thermoreversible gelation at hightemperatures as well as being non immunogenic. The high stability of thehelices should create high tensile strength in fibres or membranesformulated from CLP. These chain properties should allow the creation ofhydrogel colloids in aqueous solutions which when applied to hardsubstrates should act as soft coatings.” A suggestion is then given of asoft coating material with a ligand for a cellular receptor. Thesequence GLPGPKGDRGDAGPKGADGSP (SEQ ID NO:1) was to be added to the CLPmonomer and an example of a construct to be expressed from E. coli isprovided. With regard to this composition it is disclosed “The subjectcompositions may find use as wound dressings, allowing forneovascularisation, eye applications, matrices for artificial organs andthe like.” The combination of CLP with other repetitive functional unitsthereby combining functions is also suggested. However no examples areprovided of sequences used.

The only examples provided show a recombinantly produced synthetic CLPpolypeptide (SEQ ID NO:2) [[GAP(GPP)3]2-[GPVGSP]n with N-terminal and Cterminal spacers. The spacers are 33 amino acids and 25 amino acids inlength. Thus the repetitive GPP portion of the polymer which is 24 aminoacids in length is separated by 33+25+6 amino acids. In this manner Ecoli apparently managed to express a CLP protein of 760 amino acids,i.e. MW 63.800. The cell binding CLP had the same basic structure butthe hexamer was replaced by the cell binding sequence given aboveresulting in an amino acid length of 814 amino acids and a MW of 70.560.The repetitive GXY motif that is expressed is short and is separated bylong none repetitive sequences. The spacer DNA encodes 2 cysteineresidues and also 3 methionine residues.

The cited document states in the introductory part concerning collagen“Chemically hydrolysed natural collagen can be denatured and renaturedby heating and cooling to produce gelatin which is used in photographicand medical applications among other applications. The chain property ofcollagen responsible for this phenomenon is its ability to spontaneouslyform interchain aggregates having a conformation designated as a triplehelix” It is thus particularly remarked in this prior art document thathelical structure was required. The subsequent text is actually silenton any photographic applications and is clearly directed at completelyother matters. The subsequent text is also silent on actual degree ofexpression obtained by E. coli. The repetitive structure is present tosuch a low degree it is unlikely to retain sufficient collagen likeactivity to be useful in photographic application. In addition thepresence of cysteine and methionine residues at the levels providedherein in the expression product in fact render such inappropriate foruse in AgX emulsions for photographic applications. Furthermore it isunclear whether the use of the less repetitive sequences as describedhere actually provided any improved level of expression in E. coli. Thusa person skilled in the art of photographic applications would bedissuaded from applying the teaching of this document in photographicapplications. Firstly because it is unclear whether industrial scaleproduction would be feasible. Considering instability of repetitivesequences this is unlikely. Secondly it is unlikely due to the undesiredpresence of cysteine and methionine in AgX emulsions for photographicapplications. Thirdly this is unlikely due to the absence of helicalstructure of the expression product. The impact thereof is totallyunpredictable vis a vis stability of expression product and vis avisapplicability in photography considering the major structural differenceof current gelatins.

The same inventors as the preceding cited patent application disclose inWO93/10154 high molecular weight collagen like protein polymers havingrepetitive triads with reduced proline content. They are stated as beingcapable of production in unicellular microorganisms at high molecularweights and at high efficiency. They indicate “The uniqueness ofcollagen repetitive tripeptide is a challenge for recombinant technologyin light of the high repetitiveness of the sequence and the frequentutility of the amino acids glycine and proline in the composition. Genesencoding proteins with high levels of glycine and proline are bynecessity composed of high levels of the nucleotides guanidine andcytidine in self complementary sequences. Thus as one synthesizes thegene there is substantial opportunity for strands to loop out, singlestranded DNA to be excised, recombination events to occur which canresult in loss of segments of the gene and inefficient transcriptionand/or translation. Thus there is substantial interest in developingtechniques and compositions which provide the advantageous properties ofcollagen while at the same time allowing for stable expression of highmolecular weight collagen like proteins.” In addition it is stated. “Thepolymers will further be characterised in, being like collagen,providing helices, capable of denaturation and renaturation, forminggels etc.” A molecular weight between 30-150 kD is suggested and atleast 45 number % of the amino acids between the glycines are proline,at least 80 weight % of triads have glycine as first amino acid, atleast 40% by number of the triads comprise at least one proline. Theexample shows use of 3 types of repetitive GXO encoding sequences and Nterminal and C terminal spacer sequences. The same spacer sequences asin the previous patent application were used. The structure of therepetitive sequences was (SEQ ID NO:3)[[GAHGPAGPK]2(GAPGPAGPP)24(GAHGPAGPK)2]2=[[C]2[A]24[C]2]2. The length ofthe polypeptide produced was 561 amino acids with a MW of 46.409 Dalton.In another example the repetitive sequence was (SEQ ID NO:4)[[GAHGPAGPK]2(GAPGPAGPP)12(GAHGPAGPK)2]5=[[C]2[A]12[C]2]5. The length ofthe polypeptide produced was 777 amino acids with a MW of 64.094 Dalton,with an observed protein band at 100 kD. In the third example thestructure was (SEQ ID NO:5)[[GAHGPAGPK]2(GAPGPAGPPGSRDPGPP)12(GAHGPAGPK)2]4=[[C]2[AB]12[C]2]4. Theexample had 1065 amino acids and MW 91,966 with a protein band visual at135 kD. Apparently smaller versions were also produced with protein bandweights of 28 kD, 64 and 98 kD. With regard to expression the onlydetails provided are that detection by western blot with antisera wascarried out and that the expression of the full length polymer decreasedwith gene size, whilst the synthesis of full length mRNA was atequivalent levels. Another group of polymers with two other differentrepetitive units were produced [[C]2[DB]12[C]2]4, (SEQ ID NO:6)[[C]2[DB]6[C]2]4 (SEQ ID NO:7) and [[C]2[D]24[C]2]4 (SEQ ID NO:8),wherein B and C are as above and D=GAQGPAGPG (SEQ ID NO:9). Respectivelythese 3 proteins illustrated had 1065 amino acids and MW 91,533 D, 633amino acids and MW 55,228 D, 1065 amino acids and MW 85,386 D with aprotein band visual at 140 kD. Of the examples the only informationconcerning characteristics of the product are provided for number 6.This product is extremely soluble in water. At room temperature or abovesolutions thereof of more than 8% in water are viscous but they arefluid and form to a solid gel upon chilling to 0° C. Upon heating above28° C. the gel forms a thick solution. A thermoreversible transitionbetween liquid and gel is thus illustrated. The final example concerneda structure (GAPSQGAPGLQ)68 (SEQ ID NO:10) also with the same spacersand 1077 amino acids and a MW 91.266 D. With regard to application ofsuch polypeptide nothing more is stated than in the previously citedapplication of these inventors. Apparently by varying the blockcopolymer structure of the repetitive GXO motif it has become possiblefor expression of longer repetitive sequences to occur. How efficientsuch protein is expressed is however not clear. Yet again the expressionproblem due to repetitivity is not illustrated as being solved. It isquestionable industrial scale expression could be achieved. No teachingspecifically concerning photographic applications is provided. Inparticular all examples use spacer with cysteine and methionine which isundesirable in photographic applications. Thus a person skilled in theart of photographic applications would be dissuaded from applying theteaching of this document in photographic applications.

French patent 2685347 discusses the desirability of producingrecombinant material similar in properties to gelatin. The advantagewould be a more homogenous product solving reproducibility problems andthe chance to modify chemical functions thereof. The idea is to produceoligopeptides as gelatin substitutes. The microorganism selected was E.coli and it is stated the absence of post translation modifications suchas glycolisation common to coli is no problem. Other hosts are said tobe possible. No examples are however given of such possible hosts. Thenucleic acid sequence to be applied must comprise a gelatin peptideencoding sequence for (Gly-X-Y)n linked to Met-Cys-His-His-His-Leu-Met(SEQ ID NO:11) codons in order for selection to occur. The sequencegiven by way of example encodes (SEQ ID NO:12)Gly-Pro-Ala-Gly-Glu-Arg-Gly-Pro-Lys-Gly-Trp-Met. In a later thesis bythe inventor it became apparent that the degree of expression was infact found to be inadequate for any kind of industrial application. Inaddition the retrieval process of the produced amino acid wascomplicated. E coli was the host cell described and obviously presentedas a matter of fact the disadvantages already presented above for aperson skilled in the art interested in industrial scale production i.e.lack of secretion, instability of repetitive sequences and thus a lowdegree of expression. Finally nothing was actually illustratedconcerning application of the suggested collagen in a photographic filmin this prior art document. Thus a person skilled in the art ofphotographic applications would be dissuaded from applying the teachingof this document in photographic applications due to unpredictableoutcome of such structurally different material.

Finally a U.S. Pat. No. 5,580,712 issued to Eastman-Kodak in 1996concerned with specifically modified collagen like polypeptides andapplication thereof for photographic purposes describes that collagenlike peptizers with silver binding strengths below 50 mV can lead to ahigh degree of thin tabular grain. The document illustrates this for anumber of synthetically produced polypeptides with a length of 25 aminoacids. The document also mentions one polypeptide with a collagen likestructure was produced using recombinant technology. The recombinantpolypeptide is a synthetic polypeptide of block copolymer structureconsisting of merely 4 different amino acids. No actual expressiondetails are provided for this recombinant polypeptide, a reference ismerely made to standard molecular biology production protocols and theuse of Saccharomyces cerevisiae as expression host. The molecular weightis approximately 26 kDa. It thus is questionable whether a molecularbiologist familiar with the expression problems provided in detail inother documents of the same date and later would seriously contemplatesuch production. In addition no details concerning binding strength ofthe recombinantly produced product are provided thus it is alsoquestionable whether a person skilled in the art of photographicapplications would seriously consider use of this product in a silverhalide emulsion for photographic product or would seriously expect it toexhibit the characteristics of the short synthetic polypeptidesdisclosed. The document also suggests that the specific polypeptidesdisclosed comprising histidine and methionine at specific points namelyat Xaa of the following formula will exhibit high binding strengths andwill exhibit non tabular grain formation. The formula of the compound is(SEQ ID NO:13) Gly Pro Xaa1 Gly Leu Xaa2 Gly Pro Arg Gly Pro Pro Gly AlaSer Gly Ala Pro Gly Phe Gln Gly. Analysis of the Table providing detailsof the compounds researched by Eastman-Kodak reveals that all compoundswith high binding strengths had at least one reducing amino acid(=histidine or methionine) per 25 amino acids thus resulting in contentshigher than 400 micromoles of methionine per gram of polypeptide. Suchcompounds will not be useful in nucleation/growth AgX-emulsion processesfor photographic applications. The synthetic compounds illustrated ashaving low binding strength and favouring tabular grain formation didnot comprise any reducing amino acids Met or His. A number of other USpatents are issued to Eastman-Kodak on related subject matter. Thesepatents (U.S. Pat. No. 5,580,712 and U.S. Pat. No. 5,670,616) revealedother synthetic fragments purported to be useful for tabular grainformation but the same single recombinant product example is describedso these patents offer nothing new vis a vis recombinant collagenexpression and application of recombinant collagen in photographicapplications with AgE-emulsions. It is also derivable from thesedescriptions of Eastman-Kodak that expression of the specific sequenceshown which occurred in the host S. cerevisiae was in fact very low.Using 20 litres of culture merely ca 600 mgr of product could beisolated. No attention is paid to this aspect in the descriptionshowever. In fact the information derivable from these descriptions woulddissuade the skilled person from using this system for producingcollagen. As addressed already above this low degree of expression couldbe due to the repetitivity of the sequence to be transcribed/translatedand/or the presence of protease. In particular due to the open structureof non helical collagen any non helical collagen expression product islikely to be extremely susceptible to protease attack.

DESCRIPTION OF THE INVENTION

The subject invention is directed at use of recombinant DNA technologythus now finally enabling production of large amounts of substantiallypure collagen material overcoming the above mentioned difficulties. Forthe first time a recombinant collagen production process has beenexecuted providing high level of expression without requiring expensivemedia, expression hosts or non secreting expression hosts. In additionit has now become possible to produce collagen selected and/or adaptedfor optimal use in each particular stage of the production process ofthe photographic product where gelatin has to date been applied. Therebyrendering even further improvement possible. Also an improved AgXemulsion production process is now possible leading to a reduction inproduction costs.

The subject inventors were interested in obtaining a more uniformphotographic AgX-emulsion material and decided to investigate thepossibility of producing collagen comparable to that of collagen derivedfrom animal sources currently used in the industrial photographic paperand film making process. They expected that the use of recombinantlyproduced collagen could lead to improvements in the silver halideemulsion production due to the more uniform nature vis a vis the naturalsource which comprises a large number of contaminants and a mixture ofcollagen types of non defined and variable nature with collagen type Ias major component. The idea was to use a substance coming close to thenatural collagen rather than use of newly designed collagen likepolypeptides with highly respective block copolymer structure. The hopewas that expression problems for a sequence corresponding to the nativesequence would be smaller than those encountered in the syntheticallydesigned collagen like polypeptide sequences as described in detail inthe prior art. This could be hoped for on the basis of a lowerrepetitivity of the sequence to be expressed. On the other hand use of amore random sequence could also lead to more susceptibility to attack byvarious proteases.

However differently to the native situation for collagen we decided toabandon attempts to produce helix like structures using the recombinanttechnology. In light of page 1 of WO93,07889 which states “Unless anappropriate number of y-position prolyl residues are hydroxylated to4-hydroxyproline by prolyl 4 hydroxylase the newly synthesized chainscannot fold into a triple helical conformation at 37° C. If thehydroxylation does not occur the polypeptides remain non helical, arepoorly secreted by the cells and cannot self assemble into collagenfibrils” it could have been expected problems would arise upon applyingsecretory cells to ensure secretion. Also the potential for proteaseattack would be markedly higher due to the resulting open non foldedstructure. Thus it is surprising that our recombinant sequences resultin expression products that are quite readily secreted by the expressionhost and in high amounts. Also the production of recombinant collagenunder such conditions that the recombinant collagen compound cannot formthe fibrillar structure or the triple helix structure characteristic forthe native collagen that is currently used in photographic applicationscould also have had a questionable effect on the photographicapplication itself. In view of this difference with the currentcommercial product it was of course also questionable whether theresulting recombinant compound would be as suitable for photographicapplication as the helix comprising compound currently in use.

The results after a lot of hard work in cloning a genomic sequence forcollagen types I and III. overcoming expression problems in order toproduce sufficient amounts to start testing it in photographicapplications were however in spite of all possible setbacks unexpectedlygood. Firstly the rate of expression was unexpectedly high it was higherthan 0.95 grammes/liter and was in fact higher than 3 grammes/literoverall. This is in marked contrast to the prior art where any attemptsto express collagen or collagenlike material failed to produce more thanmilligrammes per litre it any. Thus this amazingly high rate ofexpression was considered totally unexpected firstly in view of allproblems described in the prior art. The rate of expression however evenactually outnumbered production rates achieved with expression hostPichia pastoris for other proteins. Quite specifically even thoseproteins that were not expected to be as difficult to produce in highamounts like collagen. At long last it now seems feasible a form ofrecombinant collagen could be produced in an economically interestingamount with an economically interesting expression host. Other highexpressing hosts are to be found among microorganisms of the fungaltype. In particular high expression yeasts and most specificallyprotease negative strains with low proteolytic activity are preferred.Yeasts that can be suitably used are quite specifically methylotrophicyeasts. A particularly suitable example is the yeast Pichia pastoris. Onthe basis of the criteria established as being relevant for expressionof collagen a suitable host cell capable of expression to a degree highenough and under economically feasible conditions can be found and used.

The fact that large scale production was finally made possible by thesubject inventors finally also enabled actual tests in the photographicapplication field to be carried out. Tests for photographic applicationwere only made possible after sufficient recombinant collagen had beenproduced in relatively large amounts, which is in contrast to the smallamounts required in the pharmaceutical applications described in detailin the prior art. After carrying out these tests directed atphotographic application it was discovered tabular grain formation washigh (see Table II). For the first time the application of substantiallypure collagen type III was applied in a photographic emulsion aspeptizer. The results were outstanding. Quite unexpectedly however wefound a degree of tabular grain formation higher than 80%. This evenoutdid the Eastman-Kodak polypeptide performances of polypeptides withlow birding strengths and this was considered most surprising in view ofthe fact that a binding strength of 69.5 mV was found for this productand thus in line with the Eastman-Kodak teaching non tabular grainformation was to have been expected. Thus the theory postulated byEastman-Kodak concerning the requirement of the binding strength beingbelow 50 mV to get 80% tabular grain formation is overturned. Thus thepathway was opened to develop numerous peptizers with silver bindingstrengths higher than those stated in the prior art as being suitablefor application in silver halide emulsions requiring tabular grains at alevel of more than 80%. The pathway was also opened to apply other typesof collagen than type III as major collagen component of peptizer inphotographic emulsions. Considering the degree of homology between thevarious native collagens is 40-50%, one can expect good results alsofrom slightly manipulated native sequences. An amino acid sequenceexhibiting more than 50% homology with a native collagen amino acidsequence can be expected to provide good results. Mutations of nativesequences can comprise insertions, deletions and substitutions vis a visthe native sequence. Besides, use can be made of synthetic DNA sequenceswith a certain degree of homology with native DNA sequences. Thesequences useful according to the invention must however maintain aminimum degree of variability in order to prevent expression problems oftheir encoding nucleic acid sequences. Thus the mutations should alwaysresult in an amino acid sequence with more than 4 different amino acids.The GXY motif should not be in the form of a block copolymer and shouldnot comprise spacer sequences between a number of GXY motifs. Preferablymore than 8, even more than 9 different amino acids should be present.In the example a protein with as many as 19 different amino acids isused. There are only 20 amino acids that occur naturally. Suitablybetween 10-20 amino acids or 10-19 amino acids can be used (preferablyhowever cysteine is avoided).

The invention thus comprises a tabular silver halide emulsion whereinthe tabular grains account for more than 75% of the total grainprojected area said emulsion comprising silver halide grains nucleatedin the presence of nucleation peptizer and thereafter grown in thepresence of growth peptizer, wherein at least one peptizer issubstantially pure collagen like material prepared by geneticengineering, said peptizer having an amino acid sequence comprising morethan 4 different amino acids. Such an emulsion can suitably comprise apeptizer with an amino acid sequence which exhibits more than 50%homology with native collagen, preferably more than 60% Suitably thepeptizer will have a size of at least 10 kDa. Sizes between 20-80 kDaare useful in photographic application as is apparent from the examples.Peptizers of ca 600 amino acids are illustrated.

The emulsion can comprise peptizer having an amino acid sequenceequivalent to that occurring in nature for collagen, wherein equivalentimplies amino acid identity of at least 80%, preferably at least 90%,the same as occurs in nature. The emulsion can comprise a peptizer withan amino acid sequence substantially the same as occurs in nature,wherein substantially implies mutation of less than 5 amino acids,preferably less than 3. Suitable types of collagen are I, II and III. Apreference exists for sequences close to the native sequence in order toassure activity and avoid expression problems. The DNA encoding for thepeptizer amino acid sequence can be native or synthetic. A collagen typeIII amino acid sequence according to the invention suitably comprises orhas the sequence of FIG. 3. A collagen type I amino acid sequenceaccording to the invention comprises or has the sequence of FIG. 8, 10or 12. Collagen type III has the amino acid sequence defined inreference 5 of example 1 and is incorporated by reference.

An emulsion according to the invention wherein the peptizer is presentin substantially pure form means that the peptizer is substantially freeof nucleic acid, polysaccharides and other protein. The examplesillustrate that this is indeed feasible. The presence of sugars andnucleic acid in even trace amounts could have some effects on crystalformation and it was indeed to be questioned whether sufficiently purerecombinant material for the specific photographic application could beproduced.

It is advantageous when using Pichia pastoris as expression host to usea nucleic acid sequence encoding an amino acid sequence free of thesequence MGPR (SEQ ID NO:14) even though it is present in the nativesequence of collagen type I because we have unexpectedly found thissequence is a new recognition sequence of a protease present in Pichiapastoris to which some collagen types are susceptible. It is postulatedthe protease is a Kex-2 like protease and that a Kex-2 like proteasenegative host strain will be a suitable host cell. In general terms whenusing Pichia pastoris as host cell it could be advantageous to use anucleic acid sequence encoding collagen of which the corresponding aminoacid sequence is free of [Leu-Ile-Val-Met]-Xaa-Yaa-Arg (SEQ ID NO:15)wherein Xaa and Yaa correspond to Gly and Pro or other amino acids andat least one of the amino acids between the brackets is amended. As theopen structure of non helical collagen is susceptible to proteolysis thehost should be selected and/or the sequence to be expressed ispreferably mutated or selected such that proteolysis for the specificcombination of host and sequence to be expressed is minimised. There arenumerous options open to the skilled person to realise this on the basisof common general knowledge and the subject disclosure including thecontent of the cited references.

Another way to increase the expression could lie in optimised codonusage for the host cell in which the sequence is introduced forexpression. The use of multicopy transformants is also a way in whichincreased expression can be achieved. It is suggested in the art forSaccharomyces cerevisiae expression of bovine pancreatic trypsininhibitor that the maximum level of protein secretion is ultimatelydetermined by the protein folding capacity of the endoplasmic reticulum.Exceeding this capacity by the use of multicopy transformants is thoughtto result in the accumulation of unfolded proteins in the endoplasmicreticulum and a concomitant vast decrease in the level of expression dueto physiological instability. This is described by Parekh et al in 1995(Protein Expr. Purif. 6, 537-545) and by Parekh and Wittrup in 1997(Biotechnol. Prog. 13, 117-122. It is feasible that this negative aspectis negated in the case of our recombinant collagen being expressed as anunfolded molecule and/or that this phenomenon is less relevant in otherexpression hosts in particular other yeast hosts. In yeast hosts and inbacterial hosts prolylhydroxylating mechanisms are absent and as suchexpression of collagen in such hosts will lead to unfolded collagen. Ifcollagen is unfolded it will not drain the folding capacity of theendoplasmic reticulum. Also due to outstanding solubility unfoldedunhydroxylated collagen will most likely not aggregate and accumulate inthe endoplasmic reticulum. In order to eliminate the risk of suchfolding becoming relevant to degree of expression it is thus preferredthe collagen is not hydroxylated or is at least hydroxylated to as low adegree as possible.

It has recently bean shown that it is possible to achieve hydroxylationin yeast cells such as Saccharomyces cerevisiae and Pichia pastoris bycoexpression of heterologous prolyl-4-hydroxylase. This is described byVuorela et al in 1997 (EMBO J. 16, 6702-6712) and by Vaughan et al in1998 (DNA cell Biol. 17, 511-518). As the gelling temperature of gelatinwill depend on the degree of hydroxylation it could now be possible tovary the deem of hydroxylation in a manner that will result in anexpression product with a different gelling temperature. This could beof particular interest in processes with specific temperaturerequirements that previously prohibited economical use of collagen e.g.in processes requiring temperature above room temperature to preventundesirable gelling.

The peptizer does not have to be identical to the native sequence it canbe a fragment of defined length and composition derived from a nativecollagen encoding sequence, said fragment comprising the GXY motifcharacteristic of collagen, said length being such that the fragmentweight on amino acid basis is at least 2.5 kDa. Suitably weights can bebetween 2.5 and 100 kDa. Fragments of various sizes are suitable. 5-50kDa even 20-50 kDa are suitable embodiments to be applied. The peptizercan be made de novo from a synthetic nucleic acid sequence.

Various ways of ensuring absence of helix structure are available. Forinstance ensure the peptizer is free of hydroxyproline and/or free ofprocollagen and telopeptides. Preferably for photographic applicationsthe peptizer should be free of cysteine. An AgX emulsion according tothe invention, wherein the peptizer is not deaminated is an interestingfurther embodiment of the invention as is a peptizer with an isoelectricpoint of 7-10.

Further research was carried out to ascertain what else could bediscovered to define what other parameters could be used to enhance theresults. In order to do this we analysed a number of modified collagensi.e. non recombinantly produced collagens with our recombinant collagensin tests to determine relevant parameters. We subsequently definedvarious categories of compounds as suitable for producing silver halideemulsions with the required degree of tabular grain formation.

An emulsion according to the invention in any of the other embodimentsalready mentioned with the peptizer further comprising oxidated reducingamino acids i.e. to a degree that reducing amino acids are present at alevel equivalent to a reducing strength of between 0.1-200 micromoles ofmethionine per gram of said peptize is a suitable embodiment. Preferablyless than 160 more preferably less than 120 micromoles of methionine pergram of said peptizer is present. A lower level of reducing power ispreferred so preferred emulsions according to the invention willcomprise peptizer comprising oxidated reducing amino acids to a degreethat reducing amino acids are present at a level equivalent to areducing strength of between 0, 1-80 micromoles of methionine per gramof said peptizer. Quite high levels of oxidated reducing amino acids toa degree that reducing amino acids are present at a level equivalent toa reducing strength of between 30-80 micromoles of methionine per gramof said peptizer are also able to provide adequate results. This isquite surprising considering the previous teachings concerningrequirements for low values of reducing power for tabular grainformation of numerous prior art publications. The invention also coverssuch modified collagen with lower levels of methionine than 80 μmolese.g. modified type I. The modification e.g. to the type I collagen doesnot necessarily have to be by oxidation but can also be the result ofmutation of the encoding sequence such that reducing amino acids arereplaced to the required degree by non reducing amino acids. This meansa chemical treatment step of the collagen prior to use in silver halideemulsion can be omitted with the concomitant advantages in time and costto the production process. Obviously one can apply a native collagenwhich does not have more than 80 μmoles of reducing amino acid e.g.collagen type III.

Additionally we discovered contradictory to the teaching ofEastman-Kodak mentioned earlier and published in 1996 that an emulsionaccording to the invention in any of the other embodiments mentioned,comprising a peptizer with a binding strength for silver higher than 50mV can function exceedingly well as an emulsion having a high level oftabular grain formation. A suitable peptizer will have a bindingstrength for silver below 100 mV. Contrary to the prior art teaching thepeptizer can have a binding strength for silver between 50-100 mV andprovide an emulsion with excellent tabular grain percentage.

The silver halide emulsion resulting from application of such collagenlike material will suitably exhibit more than 80% tabular rains,preferably more than 90%. Most preferred is a tabular grain percentagehigher than 95%. The grains will exhibit an average aspect ratio higherthan 5 when determined using the single jet method under the reactionconditions described in the example. Note these reaction conditions arenot the optimised reaction conditions used in actual photographicemulsion processes for obtaining the highest aspect ratios but merelyprovide an indication of whether the material is suitable to achievehigh aspect ratios when such optimised conditions are applied. Thecompounds according to the invention upon application of optimisedconditions currently used on normal collagen e.g. using the double jetmethod and an additional ripening process are expected to exhibit muchhigher aspect ratios. The applied test is merely a quick indicator ofhigh aspect ratio forming capacity and the person skilled in the artwill realise what measures can be taken to enhance the result further.

In our test the ripening process is carried out without any furtheraddition of peptizer or extra silver. Obviously in a process accordingto the invention the ripening step could comprise such further addition.The peptizer can be the same material for both nucleation and ripeningstage. An additional addition in the ripening, stage could beadvantageous due to increased steric stability at this stage.

A preferred AgX emulsion according to the invention comprises a peptizerthat is stable vis a vis silver halide tabular grain formation at a pHbetween 4-8. Conventional gelatin derived from lime bone and hydrolysedgelatin do not exhibit such good tabular grain formation results at pHhigher than 5.5. The peptizers according to the invention e.g. nativerecombinant collagen III do exhibit such characteristic thus making therequirement for strict pH control during emulsion production storage andapplication redundant. The native recombinant collagen can also undergooxidation of methionine to exhibit improved behavior. Suitably themethionine level will be less than 80 micromoles per gramme. An emulsionaccording to the invention can thus have a nucleation and growth pHbetween 4-3 without negatively affecting tabular grain formation. In theprocess of emulsion production a variation in pH will not negativelyeffect the outcome upon further processing of the resulting emulsion forphotographic element production.

An emulsion according to the invention offers the advantage overconventional collagen comprising silver halide emulsions that thepeptizer is of a homo disperse nature. The crystallisation process byvirtue of this fact is also more homogeneous with all the advantagesmentioned above. It is possible to add at various stages of thenucleation and growth of the silver halide crystals a further homogenouspeptizer also clearly defined and substantially pure thus combiningprice effectiveness and controlling crystallisation properties in aregulated manner previously not possible with a collagen like peptizer.As there are many types of collagen naturally these can also be appliedin photographic silver halide emulsions according to the invention.

There are 23 collagen genes that have been di covered so far. Most ofthese have been sequenced in part or as a w % hole. The databankscomprise the various sequences therefore. Genbank for example has thecoli sequence under accession number Genbank U08020 and the colIIIsequence is given in reference 5 of example 1. These native collagengenes exhibit homologies when compared amoung themselves of 40-50%. Therelevant information of the cited references with regard to sequencedata is hereby incorporated by reference see also e.g. WO95/31473 page5. Application of any of these native sequences as such or modified,obtained by isolation from a natural source or by chemical synthesis, inorder to produce a collagen like compound as defined above according tothe invention, said compound subsequently being used in a silver halideemulsion for photographic application is covered by the subjectinvention. Suitably the sequences are applied i.e. sequences encodingpolypeptides with the natural amino acid sequences or similar to thenatural amino acid sequences as long as the encoded polypeptide fallswithin the parameters disclosed above. The type I has been the typemostly applied in silver halide emulsions as the source for silverhalide emulsion gelatin was animal bones of which type I collagen is themost predominantly present collagen type. Now it has become possible toalso test and apply other collagen types for suitability in silverhalide emulsions. The other collagen types have not been applied as suchin the prior art in photographic applications and certainly not as suchin silver halide emulsions. Naturally a number of them have been presentin animal tissue that has been used to date. Now it has become possibleto see whether these collagen types can in fact be responsible for orcontribute to even more favorable properties to photographic productspreviously unrecognised. A photographic sensitive emulsion comprisingrecombinant collagen like polypeptide other than type I as collagen likecomponent is also considered to form a suitable embodiment of theinvention as long as the specific requirements set out above arefulfilled. Specifically application in a silver halide emulsion iscovered. In the case oz silver halide emulsions a 100% uniform source ofcollagen is expected to provide maximum homo dispersity of crystalformation. The requirement of homodispersity and the value thereof havebeen addressed elsewhere in this description of the invention. It is notnecessary for the peptizer to comprise the full length collagen it cancomprise a fragment thereof. Suitably such fragment is however at least2.5 kDa, preferably more than 10 kDa in length to ensure sufficientrandomness of expression whilst maintaining collagen aspects requiredfor the peptizer.

Besides the emulsions described above the invention is also concernedwith a process for preparing tabular silver halide emulsion wherein thetabular grains account for more than 75% of the total grain projectedarea said process comprising nucleation of silver halide grains in thepresence of nucleation peptizer and thereafter growing said silverhalide grains in the presence of growth peptizer, wherein both peptizersare present in a defined amount and at least one peptizer is collagenlike material prepared by genetic engineering, said peptize having anamino acid sequence comprising more than 4 different amino acids. Such aprocess according to the invention can comprise addition of the peptizerin the nucleation step and or during the grain growing step, saidpeptizer can be selected from any of the embodiments disclosed above orin the claims. In a special embodiment the process comprises addition ofthe peptizer both in the nucleation step and during the grain growingstep. The peptizer to be used when both steps are taken can be the sameor different depending on the circumstances of the case.

After preparing a AgX emulsion according to the invention the AgXemulsion can undergo the standard procedures for preparing aphotographic element. The emulsion can be applied in a manner known perse for achieving a silver halide emulsion layer on photographic materialwherein the silver halide crystals of said layer have an aspect ratio of5 or more.

Said photographic element is suitably a material sensitive to light,laser or x-ray radiation, said element being selected from black andwhite reversal film, black and white negative film, colour negativefilm, colour reversal film, film in which the sensitive photographiccomponents are digitally scanned, black and white reversal paper, blackand white paper, colour paper, reversal colour paper, paper in which thesensitive photographic components are sensitized by laser radiation outof a digital database. A photographic element obtained according to sucha process is also covered by the invention as well as an element usingthe direct positive process with internal sensitised silver halideemulsion and elements using heat development.

Another aspect of the invention lies in a process of producingrecombinant collagen like polypeptide comprising expression of acollagen like polypeptide encoding nucleic acid sequence by amicroorganism to a degree exceeding 0.95 grammes/liter, said recombinantcollagen being free of helix structure. Preferably the expression occursin a microorganism other than E. coli or Saccharomyces cerevisiae inorder to ensure high expression and preferably secretion. The processcan suitably be carried out with a fungal cell preferably a yeast cell.Suitably the host cell is selected from the group consisting of highexpression host cells like Hansenula, Trichoderma, Aspergillus andPichia. Fungal and particularly yeast cells are preferred to bacteria asthey are less susceptible to bad expression of repetitive sequences.Most preferably the host will not have a high level of proteases thatattack the collagen structure expressed. In this respect Pichia offersan example of a very suitable expression system. Preferably themicroorganism is free of active post translational processing mechanismfor processing collagen like sequences to fibrils thereby ensuringabsence of helix structure in the expression product. Also such aprocess can occur when the microorganism is free of active posttranslational processing mechanism for processing collagen likesequences to triple helices and/or when the nucleic acid sequence to beexpressed is free of procollagen and telopeptide encoding sequences. Thehost to be used doe not require the presence of a gene for expression ofprolyl-4-hydroxylase the enzyme required for collagen triple helixassembly contrary to previous suggestions in the art concerning collagenproduction. The selection of a suitable host cell from known industrialenzyme producing fungal host cells specifically yeast cells on the basisof the required parameters described herein rendering the host cellsuitable for expression of recombinant collagen according to theinvention suitable for photographic applications in combination withknowledge regarding the host cells and the sequence to be expressed willbe possible by a person skilled in the art.

To ensure production of a non cleaved sequence a process according tothe invention for producing recombinant collagen like material comprisesuse of a nucleic acid sequence encoding recombinant collagen amino acidsequence substantially free of protease cleavage sites of proteaseactive in the expression host cell. In the case of Pichia pastoris forexample and possibly also for other host cells a nucleic acid sequenceencoding collagen of which the corresponding amino acid sequence is freeof [Leu-Ile-Val-Met]-Xaa-Yaa-Arg (SEQ ID NO:15) wherein Xaa and Yaacorrespond to Gly and Pro or other amino acids and at least one of theamino acids between the brackets is amended could be preferred. Apreferred process according to the invention comprises use of themicroorganism Pichia pastoris as expression host.

The process suitably provides expression leading to peptide harvestexceeding 3 grammes/liter. The process can suitably be carried out withany of the recombinant collagen like peptizers defined above for theemulsion according to the invention. As is apparent from the examplesunder the circumstances described therein multicopy transformantsprovide more than 14 grams of gelatin per liter of clarified broth at abiomass wet weight of 435 grams per liter. Most suitably the productresulting from microbial expression is isolated and purified until it issubstantially free of other protein, polysaccharides and nucleic acid.As is apparent from the examples numerous methods are available to theperson skilled in the art to achieve this. The process according to theinvention can provide the expression product isolated and purified to atleast the following degree: content nucleic acid less than 100 ppm,content polysaccharides less than 5%, content other protein less than incommercial products. More preferably the DNA content of less than 1 ppm,RNA content less than 10 ppm even less than 5 ppm and polysaccharidecontent less than 1% can be achieved.

Another aspect of the invention covers novel recombinant collagen likepeptides. In particular the invention covers a substantially purecollagen like material prepared by genetic engineering of collagenencoding nucleic acid, said peptizer having an amino acid sequenceexhibiting more than 40% homology with native collagen and comprisingmore than 4 different amino acid types. The nucleic acid sequence can bederived from the native sequence or be synthetic nucleic acidsynthesized de novo. Other suitable embodiments are those peptizersdescribed in the emulsion embodiments according to the invention. Asclose a homology as possible is preferred as homologies higher than 50%preferably even in the order of 80% are desired, even 80-100%. Wespecifically point out the products are preferably free ofblockcopolymer structure within the GXY motif sequence.

in a preferred embodiment of the invention the collagen like materialcomprises no cysteine residues. The presence of cysteine in photographicproduct will disturb the product manufacturing process. It is thuspreferred that cysteine is present in as small an amount as possible.This can be achieved either through chemical modification of therecombinant product or mutation in the nucleic acid sequence encodingthe product by mutation or deletion of a cysteine encoding sequence suchthat cysteine is no longer encoded. Suitably photographic applicationswill employ material comprising less than 0.1% cysteine.

In particular for the optimal silver halide emulsion homogeneity of thecollagen material is of the utmost importance. It is not merely aquestion of absence of impurities that provides an improvement it is thepossibility of providing molecules of exactly the same composition andlength allowing good control of the extremely sensitive process ofcrystallisation and also enabling uniform crystal growth. For thisreason recombinant collagen like material will be valuable for this partof the photographic manufacturing process. In addition the absence offibril formation and even of triple helices is required for thisparticular application in the photographic manufacturing process anaspect that until now had been completely overlooked. The insight in therelevance of the number of reducing groups in the photographic materialis also of great importance. This is not the rigid low amount suggestedin the prior art required for tabular grain formation. Thus thereduction in cysteine, histidine and methionine levels in the collagenlike material to be applied forms a preferred embodiment of theinvention.

The compounds according to the invention have also revealed anadditional advantage. The known collagen materials e.g. regular andhydrolyzed collagen from animal sources such as bone and hide result inlow tabular grain formation of the photographic film emulsion at higherpH than 5.5. The new group of recombinant collagens have been found toresult in the same astonishingly hitch degree of tabular grain formationnot only at pH 5.5 but also at higher pH e.g. pH 7. This offers thepossibility of preparing silver halide emulsions which have lessstringently controlled pH as the new compounds are apparently less pHdependent than the non recombinant collagens. Thus the invention is alsodirected at recombinant collagen like compounds that can be used in theproduction of silver halide emulsions at a pH between 4-8 whilst stillarriving at high tabular grain percentages i.e. higher than 50%0preferably higher than 80% An additional characteristic of therecombinant collagens that can be considered useful is the fact that theisoelectric points are basic as opposed to the recombinant Eastman-Kodakpolypeptide described in 1996 which has an acidic IEP. It is expectedthat the fact that the recombinant collagen according to the inventionhas an amino acid composition wherein more than 4 amino acids arepresent offers increased variability in the encoding sequence and thusallows higher degree of expression. Additional variety is introduced byuse of a sequence with a GXY motif with less than 33% proline in thetotal GXY sequence. The good expression is achieved without use of ablock copolymer amino acid structure in the GXY sequence.

The invention will further be illustrated by the examples.

EXAMPLES Example 1

Gelatins, collagen or collagen fragments expressed as recombinant,heterologous protein in expression host organisms like yeast, fungibacteria for photographic applications by recombinant-DNA techniques hasseveral advantages. (i) In contrast to for example traditional gelatins,recombinant molecules can be produced as rigorouslv non cross-linked.(ii) The molecular composition is precisely-defined. (iii) The moleculesproduced are of a single type (or a well-defined mixture of only a fewmolecules), with minor or negligible contamination from otherproteinaceous or non-proteinaceous molecules. The molecular weightdistribution is very: narrow and mono-disperse (single-componentgelatins) or oligo-disperse. (iv) The product can be manufactured in ahighly reproducible way, i.e. with constant quality. Especially yeastare well-suited production organisms for such polypeptides with a highlyrepetitive, glycine- and proline-rich sequence.

Whereas these molecular features often cause genetic instability (e.g.recombination and shuffling of parts of the gene) in bacterial systems,this appeared to be not much of a problem in yeast [1,2]. They areeukaryotic cells in which post translational modifications likehydroxylation can be effectuated, and which allow to choose for eitherefficient secretion or intracellular expression. Several species growefficiently on cheap substrates like methanol, in contrast to animalcell cultures. Secreted production allows efficient recover, of theproduct during or after fermentation (contrast with plants). Severalstrong and tightly-regulated inducible promoters are available for yeastsystems, allowing a highly efficient expression and minimizing possiblenegative effects on the viability and growth of the host cells. As oneof several well-suited systems that are available, we have chosen forsecreted production by the methylotrophic yeast Pichia pastoris. Ourexpression levels are among the highest ever reported for recombinantproteins, indicating the ability of this expression host to cope withthe aforementioned or the structure of gelatin collagen at genetic (DNA,RNA) and protein levels. After transformation of the host, theintegrative is incorporated into the yeast's genome, resulting ingenetical stability of the transformants (loss of plasmids is then of noimportance). It is possible to generate transformants (with theheterologous target gene under the control of e.g. the alcohol oxidase(AOX) promotor), in which the recombinant gene is either incorporatedinto the HIS4 locus or the AOX1 locus. In the latter case, depending onthe type of integration, the AOX1-gene is disrupted, leading to slowutilisation of (and slow growth on) methanol (Mut^(s) phenotpe). If thefunctional AOX1 gene is still present, the phenotype is Mut⁻. Althoughboth phenotypes can be used, we generally preferred fast growth andthus, our protocols were mainly directed at the generation and selectionof Mut⁻ transformants. It is self-evident that yeast or fungalexpression systems other than the P. pastoris expression system could inprinciple be used equally well for the efficient production ofrecombinant gelatins, depending on the exact type and quality ofmolecule to be produced, on the production scale envisaged, and on theproduction costs and applicable market prices. The Pichia system wasused as a fast and efficient system for pilot production and relativelyeasy pro-duct recovery.

Materials, Methods & Analyses General Molecular-Biological Techniques

Cloning procedures were carried out essentially according to Maniatis etal. [3]. DNA was isolated using Wizard Plus SV miniprep, or Qiagenmidiprep systems. DNA was isolated from agarose gels using the QIAquickGel Extraction Kit (Qiagen). All enzymes used were from Pharmacia unlessotherwise stated and were used according to the manufacturer'srecommendations. Transformation of E. coli was performed by standardelectroporation using the BioRad GenePulser. All procedures involvingthe handling and transformation of Pichia pastoris and the expression ofproteins in this host organism were essentially carried out according tothe manual of the Pichia Expression Kit (from Invitrogen) [4].

Insertion of a Rat COL3A1 cDNA Fragment into a Yeast (Pichia pastoris)Expression Vector

Plasmid pRGR5, containing a partial rat proα1(III) collagen cDNA, was akind gift of Dr. Vuorio [5]. It was digested with PstI, yielding anapproximately 0.7 kb fragment of the helical domain. Using the 3′-5′exonuclease activity of T4 DNA polymerase the fragment was blunt-endedand subsequently ligated with T4 DNA ligase to SnaBI digested and CIPdephosphorylated pPIC9 Pichia pastoris expression vector (Invitrogen).The ligation reaction was then used to transform E. coli JM109.

It will be understood that the choice of possible and suitable vectorsis not restricted to pPIC9. Anyone skilled in the art will be able touse and adapt a number of other possible vectors such as pHIL-S1, inwhich a Pichia pastoris acid phosphatase 1 (Pho1)-signal instead of theSaccharomyces cerevisiae-derived alpha-mating factor (αMF) prepro signalis used, or pHIL-D1, for intracellular expression, and many others.

Plasmid DNA was isolated, and the sequence of the pCOL3A1 construct thuscreated (FIG. 1) was verified by sequencing according to the method ofSanger [6], using an automated sequencer (ALF DNA Sequencer, Pharmacia)and by using the 5′AOX1, 3′AOX1 and α-Factor (αMF) sequencing primerssuggested in the Pichia Expression Kit (see FIG. 2) (SEQ ID NOs:35-37),respectively. The protein sequence expected for the expressed protein isindicated in FIG. 3 (SEQ ID NO:38).

Transformation of Pichia pastoris with pCOL3A1

In order to obtain Mut⁻ transformants upon transformation of Pichiapastoris, the construct was linearized with SalI. In order to obtainMut^(s) transformants the construct was digested with BglII. Afterphenol extraction and ethanol precipitation, the con-struct was thenused to transform Pichia pastoris strain GS115 (Invitrogen) usingelectroporation according to Becker and Guarente [7] using the BioRadGenePulser (set at 1500V, 25 μF and 200Ω and using 0.2 cm cuvettes). Thetransformation mix was plated out on Minimal Dextrose plates (MD-plates;1.34% YNB, 4×10⁻⁵% biotin, 1% dextrose and 1.5% agar) in order to selectfor the presence of the vector which converts the His⁻ strain GS115 toHis⁻. After growth at 30° C. for 3 days, several colonies were selectedfor PCR confirmation of the Mut genotype. Genomic DNA was isolatedaccording to the yeast miniprep method of Lee [8] and RNase A treated.PCR was performed using 100 ng of genomic DNA. 50 pmol 5′AOX1 primer, 50pmol 3′AOX1 primer, 1.25U Taq polymerase (Pharmacia), 0.2 mM dNTPs(Pharmacia) and 1× Taq buffer (Pharmacia) in a total volume of 50 μl.After an initial denaturation at 94° C. for 5 minutes. 30 cycles wereperformed consisting of 1 minute at 94° C., 1 minute at 57° C. and 2minutes at 72° C. Final extension was at 72° C. for 10 minutes. The PCRmachine used was the Perkin-Elmer GeneAmp 480. Agarose gelelectrophoresis should reveal a 2.2 kb endogenous AOX1 band for Mut⁻transformants. Transformants without 2.2 kb band are Mut^(s). Verifiedtransformants of both the Mut⁻ and Mut^(s) genotype were selected forsmall-scale expression in 50 ml conical tubes (placed at an angle andwith the cap loosely attached), or in 100 ml or 11 (baffled) flasks.

Expression of COL3A1 Fragment

Expression was performed essentially as described in the PichiaExpression Kit manual. Briefly, transformants were grows overnight inBMG (100 mM potassium phosphate pH6.0, 1.34% YNB, 4×10⁻⁵% biotin and 1%glyceol) to an OD₆₀₀=2−6. Cultures were then centrifuged and resuspendedin BMM (as BMG but 0.5% methanol replaced the glycerol) to an OD₆₀₀ of1.0. Cells were grown for 4 days at 30° C. and 250 rpm, with methanolbeing added to 0.5% every day. 10 μl of the culture supernatants wasanalyzed by SDS-PAGE according to Laemmii [9] in a BioRad mini-PROTEANII system. Coomassie Brilliant Blue staining revealed several bands, thehighest of which had the expected apparent length of about 29 kD. Itshould be noted that gelatin, collagen and collagen fragments migrateaccording to an apparent Mw, which is about a factor 1.4 higher than thetrue Mw, at least partly due to the relatively low mean residue Mw [12].

In order to establish their identity, an SDS-PAGE gel loaded withacetone-fractionated COL3A1 fermentation supernatant (see below for thefractionation procedure) was blotted to an Immobilon P^(SQ) membrane(Millipore) using the Biorad Mini Trans-blot Cell. Quantitative transferwas achieved by applying 100V for one hour, using CAPS buffer (2.2 gCAPS per liter of 10% MeOH, pH 11). After staining with CoomassieBrilliant Blue, the four most prominent bands were cut out and theN-terminal sequence was determined by Edman degradation. The 29 kD banddid not give any signal and is probably N-terminally blocked. One of thetwo smaller fragments was not pure enough to be sequenced. The other twosmaller bands did give readable signals and are underlined in FIG. 3. Itis clear that the bands are caused by some form of proteolysis, whichcan be explained by the fact that gelatin is a very open protein in therandom coil conformation and is thus highly susceptible to proteolysis.

Protease Activity

Degradation of the collagen was observed of collagen types I and IIIduring fermentation at pH 5.0. Tests were carried out to furthercharacterize this degradation. This degradation was markedly reducedwhen carrying out the fermentation process at a lower pH. SpecificallypH 3.0 provided good results. We also researched the effect of additionof casamino acids. The addition gave protection for both types ofcollagen at pH 5.0. Furthermore the addition provided even betterprotection for collagen type I also at pH 3.0. This additionalprotection was not noticeable in the case of collagen type III at pH3.0. It is presumed extracellular neutral proteases attack the collagenwhich is extremely vulnerable to proteolytic degradation resulting fromit's random coil conformation. (See below for a description of thefermentation procedure). The tests were carried out with thecollagen-containing supernatants of a pCOL3A1 fermentation at pH 3.0,where degradation during fermentation was minimal. After removal of thecells by centrifugation, the pH of the supernatants was adjusted to pH5.0. Subsequently, parallel incubations were carried out with thefollowing additions:

(1) fresh Pichia pastoris cells (washed with MilliQ)(2) fresh Pichia pastoris cells (washed with MilliQ) and glass beads:this mixture was vortexed (positive control I).(3) nothing added (negative control)(4) trypsin (5 mg/ml) (positive control II).

All samples were incubated for 96 hours at 30° C. and pH 5.0. (Thesewere the conditions that caused degradation of the gelatin duringfermentation). Finally, the incubated samples were analyzed on anSDS-PAGE gel according to Laemmli [9] in a Biorad mini-PROTEAN IIsystem, followed by a Coomassie Brilliant Blue staining.

The results were:

(1) incubation at pH 5.0 with washed intact cells caused degradation ofpCOL3A1 (originally produced at pH 3.0) into 4-5 discrete bands,probably as a result of cell-surface associated proteolytic enzymeactivity:(2) addition of broken cells caused degradation of both collagen typesinto a large number of proteolytic products (positive control I);(3) no degradation occurred in the absence of cells at pH 5.0;(4) addition of trypsin caused massive degradation of the gelatin(positive control II).

In different experiments, we verified that after removal of the cells atthe end of the fermentations, the recombinant gelatins in the cell-freefermentation broth were stable for several days in the temperature rangeof 0-30° C. and the pH range of 3.0-7.0. Thus, some proteolyticdegradation of gelatin occurred during fermentation, but after removalof cells, no relevant proteolytic activity remains and, no furtherprecautions are necessary to stabilize the product. A similar stabilitywas observed for the COL1A1 products described below. This stability ofthe recombinant gelatins came as a surprise, as they are nothydroxylated (shown by analysis of amino acid composition, as describedbelow) and, accordingly, non-helical, i.e. without any secondarystructure. The total absence of secondary structure (i.e. ofcollagen-type helix) and of hydroxyproline was verified, respectively,by circular dichroic spectroscopy (CD) according to ref. [13] and byHPLC analysis of the amino acid composition after full hydrolysis of thepeptide bonds. At 5 degrees Celsius it was ascertained that theexpression product remained largely in the random coil configuration andis thus essentially non gelling. This is in accordance with the absenceof helix stabilizing hydroxyprolines as confirmed by the experiments.The recombinant gelatins are thus extremely open molecules (and as suchunparalleled polypeptides!) that are Sound to be extremely prone toproteolytic degradation. The unexpected stability of the pro-duct inthis expression host (also after secretion) greatly facilitates thedownstream processing and isolation of the product from this expressionsystem and obviates the repeated addition of expensive and instableinhibitors of proteolytic activity (e.g. para-methyl-sulfonyl fluoride(PMSF)). In addition, it opens up possibilities for minimizing gelatindegradation during high cell-density fermentation, viz. by continuouslyseparating the product from the cells during fermentation, using simplemicro filtration or dialysis against nutrient broth, and byrecirculating the cells to the fermenter Previously only TRIPLE-HELICALcollagen or folded polypeptides have been produced. These are moreresistant to proteases. Triple helical collagen is even fully resistantto trypsin, pepsin, and other well-known proteases. In contrast, theproduction of intact, non-hydroxylated and unfolded gelatin was thusexpected to be extremely difficult.

Production in a Protease-Deficient Strain

In order to investigate if the pep4 proteinase A deficient strainSMD1168 (Invitrogen), is better suited for the expression of theprotease sensitive gelatin sequences, this strain was also transformedwith the pCOL3A1 construct. Methodology was as described above.Unfortunately there was no clear positive effect in both shake flask andfermenter expression experiments.

Analysis of Glycosylation

In order to establish if the protein is glycosylated, a PAS staining,involving the application of Schiff's reagent after oxidation byperiodic acid, was performed on an SDS-PAGE gel. The gel was incubatedfor 1 hour in 12.5% TCA. 1 hour in 1% periodic acid/3% acetic acid, 1hour in 15% acetic acid (replaced every 10 minutes) and 1 hour inSchiff's reagent (at 4° C. in the dark). The gel was then Washed twotimes for 5 minutes in 0.5% sodium bisulfite and destained in 7% aceticacid. The expressed protein bands gave no signal, while there was asignal from a positive control (carboxypeptidase Y). As expected, nosignal was obtained with a negative control (E. coli extract). It can beconcluded that the expressed protein is not glycosylated.

Northern Blotting

A northern blot of methanol grown cells was performed. RNA was isolatedaccording to the method of Schmitt et al. [10]. The PCOL3A1 vector wasdigested with Eco-RO/SphI to give a 0.5 kb COL3A1 fragment. The fragmentwas ³²P random primer-labeled and hybridized to the blot, after whichthe blot was washed to a final stringency of 0.2×SSC at 65° C.Autoradiography revealed a messenger of the expected length (1.3 kb).

Pichia Transformants Containing Multiple Copies of the HeterologousCOL3A1 Gene

In order to investigate whether gelatin expression levels can beenhanced even further, the G418 multi copy selection method of Scorer etal. [11] has been applied. The pPIC9K vector was digested With BamHIEcoRI and the 9.0 kb band was isolated. The pCOL3A1 vector was alsodigested with BamHI/EcoRI and the resulting 1.0 kb fragment was ligatedto the 9.0 kb pPIC9K band after which E. coli JM109 was transformed. Theconstruct pCOL3A1K (FIG. 4) thus obtained was verified by restrictiondigestion.

Pichia pastoris GS115 was transformed with the pCOL3A1K vector asdescribed before (digested with SalI in order to obtain Mut⁻transformants). In order to select for multicopy transformants, the his⁻colonies on MD-plates were pooled (approximately 6000) and subjected tosecondary screening on plates containing a series of 10 different G418(Gibco-BRL) concentrations ranging from 0.25 to 4.0 m/z/ml. The cellswere plated at a density or approximately 10⁵ cells per plate. Afterincubation for 4 days at 30° C. several resistant colonies of each G418concentration were transferred to fresh plates at the correspondinglevel of G418 to verify their resistance.

In order to determine the copy number of the pCOL3A1K vector in the G418resistant transformants, a semi-quantitative dot blot was performed.Genomic DNA of the verified G418 resistant transformants was isolatedaccording to the protocol of Lee [8] and RNase A treated. Approximately200 ng of genomic DNA of each of 40 transformants (4 per concentrationof G418) was transferred to a positively charged nylon membrane(Boehringer Mannheim) by means of a vacuum blotting device (Gibco-BRLConvertible system). As a I-copy control a pCOL3A1 transformant whichhad been verified to contain only 1-copy by Southern blot was alsotransferred to the blot (in duplo), as well as a non-transformed control(in duplo).

The pCOL3A1 vector was digested with EcoRI SphI to give a 0.5 kb COL3A1fragment. This fragment was ³²P random primer-labeled and hybridized tothe dot blot filter. After washing to a final stringency of 0.1×SSC at65° C. autoradiography was performed. After stripping (the efficiency ofwhich has been checked), the membrane was hybridized to a probe derivedfrom a verified Pichia pastoris URA3-fragment, which had been picked upby PCR with heterologous URA3 primers. This control serves fornormalization of the COL3A1 signals for the amount of DINA loaded. Themembrane was washed and subjected to autoradiograms as described for theCOL-3A1 probe. The signals on both autoradiograms were densitometricallyquantified using a gel scanner (PDI, Pharmacia). As expected there wasno COL3A1 signal for the 0-copy controls, while there was a URA3 signal.The copy number can be estimated by calculating the ratio of COL3A1signal for each transformant and the average COL3A1 signal obtained forthe 1-copy controls, as normalized by the ratio of the respective URA3signals (i.e. to account for differences in the amount of DNA blotted tothe membrane). Transformants containing approximately 1 to 15 copieswere thus obtained.

Expression of COL3A1 Fragments in Multi-Copy Transformants.

A series of transformants containing 1 to 15 copies was subjected tosmall-scale expression as described above. Since SDS-PAGE indicated ahigher yield at higher copy number, further tests were carried out at a100 mL scale with the 2-, 10- and 15-copy transformants. They were grownovernight in 100 ml flasks, containing 25 ml BMG (100 ml potassiumphosphate pH 6.0, 1.34% YNB, 4.10⁻⁵% biotin and 1% glycerol). Aftercentrifugation at 1500-3000 g for 5 minutes, the cells were resuspendedin 100 ml BMM (as BMG but 0.5% methanol instead of the glycerol). Theywere grown in 1 liter baffled flasks at 30° C. and 250 rpm for 4 days,with methanol being added to 0.5% every day. 1 ml samples were takeneach day and analyzed on SDS-PAGE. A higher copy number resulted in ahigher amount of gelatin product. Selected 5- and 15-copy transformantswere used for expression tests in a fermenter at a 1 L-scale. Thehighest COL3A1 production was obtained with the 15-copy transformant(about 14.8 g gelatin/L in the extracellular medium at a dry biomass of177 g/L and after about 184 hours of fermentation, i.e. about 7.7 g/Loverall, or 42 mg/(L.hour); at a dry biomass of 110 g/L and after about120 hours of fermentation, it was about 7 g gelatin/L in theextracellular medium, i.e. about 3.7 g/L overall, or 31 mg/(L.hour)).

Cloning of a Mouse COL1A1 fragment (COL1A1-1) Mouse

Primers were designed on the known sequence (FIG. 5) (SEQ ID NOs:39-46,respectively). PCR was performed on Mouse 17-day Embryo QUICK-Clone™(fibroblast) cDNA (Clontech), using 0.4 ng of cDNA, 0.4 μM C1A1-FWprimer (FIG. 5), 0.4 μM C1A1-RV1 primer (FIG. 5), 1× Advantage KlenTaqPolymerase Mix (Clontech), 0.2 mM dNTP's (Pharmacia) and 1× KlenTaq PCRreaction buffer (Clontech) in a total volume of 20 μl. After an initialdenaturation at 94° C. for 4 minutes, 35 cycles were performed of 1minute at 94° C., 1 minute at 68° C. and 2 minutes at 72° C. Finalextension was at 72° C. for 10 minutes. Agarose gel electrophoresisshows a 1 kb band, which is the size predicted from the sequence. DNAwas isolated from the agarose gel and subsequently digested with NcoIand XhoI restriction enzymes. The digested fragment was isolated fromagarose gel and cloned into the Pichia pastoris expression vector pPIC9according to the following strategy (FIG. 6). First, an adaptorcontaining a NcoI and a XhoI site, was inserted in the multiple cloningsite of pPIC9, yielding pPIC9*. The adapter was prepared by annealingthe synthetic oligonucleotides N—X-FW and N—X-RV as shown in FIG. 5 andFIG. 6. The single-strand overhang originating from the 5′ end of theoligonucleotide N—X-RV was designed to form an EcoR I site afterannealing with the EcoR I-digested vector. The 5′ overhang from N—X-FW(Xho I*) was complementary to the overhang created by the action of XhoI on the vector, but did not give rise to an Xho I site after ligation.Because the target vector, pPIC9, has an Nco I site outside the multiplecloning site, pUC18 has been used as an intermediate vector for thecloning of this fragment. The section between BamHI and EcoRI of thealtered multiple cloning site of pPIC9* was transferred to the multiplecloning site of pUC18 vector, resulting in pUC18*. The NcoI-XhoIdigested fragment COL1A1-1 was ligated in the pUC18* vector between theNcoI and XhoI sites. From this pUC18-COL1A1-1 construct the COL1A1-1fragment, together with the part of the multiple cloning site from thepPIC9, was digested with BamHI and EcoRI and ligated in pPIC9, yieldingthe construct pCOL1A1-1 (FIG. 7). Thus, a partial NcoI-digestion of thepPIC9 was not necessary. The correct insertion in pPIC9 was firstchecked by restriction analysis and then by DNA sequencing.

Transformation of Pichia with pCOL1A1-1 and Expression of the COL1A1-1Fragment

Pichia pastoris GS115 was transformed with the pCOL1A1-1 vector asdescribed for the pCOL3A1 vector. Sal I-digested DNA was used in orderto specifically generate Mut⁺ transformants. Several transformants wereused for small-scale expression in shaking flasks and one of those wasselected for expression in the fermenter at a 1-100 L scale. Typicalyields are in the range of 4-5 g gelatin/L in the (extracellular) medium(as determined after acetone fractionation, described below), at a drybiomass of 100-120 g/L (about 3 g gelatin/L overall). The target gelatin(FIG. 8) (SEQ ID NO:47) has a theoretical Mw of 27.4 kD. Collagenousproteins and gelatin are known to migrate at an apparent Mwapproximately 1.4 times higher than the true Mw [10]. In agreement, anSDS-PAGE band migrating at an apparent Mw of about 38 kD was observed(interpolated value obtained with globular protein Mw markers). Inaddition, three shorter products with an apparent Mw of 24, 18 and 15 kDwere observed (interpolated values). These could be the result of earlyproteolytic activity in the intracellular, cell surface-associated orextracellular compartments, or from problems at the level oftranslation. The degradation products were present already at very earlystages of induction and no further degradation occurred. Even incubationin the presence of washed intact cells at pH 5.0 and 30° C. during 96hours did not cause further degradation of pCOL1A1-1 with respect to thesituation after fermentation at pH 3.0. (Massive degradation occurred inthe presence of trypsin, as a positive control). In order to verify thatproblems at the mRNA level were not responsible for the occurrence ofthe 24, 18 and 15 kD products, Northern blotting was performed asdescribed for COL3A1, using a ³²P random-primer labeled 1.0 kb NcoI/XhoICOL1A1-1 fragment from pCOL1A1-1 as the probe. The expected 1.6 kbmessenger was found.

In order to establish the identity of the observed fragments, anSDS-PAGE gel loaded with acetone-fractionated COL1A1-1 fermentationsupernatant was blotted to an Immobilon P^(SQ) membrane (Millipore)using the Biorad Mini Trans-blot Cell. (See below for a description ofthe acetone fractionation procedure). Quantitative transfer was achievedby applying 100V for one hour, using CAPS buffer (2.2 g CAPS per literof 10% MeOH, pH 11). After staining with Coomassie Brilliant Blue, thefour most prominent bands were cut out and the N-terminal sequence wasdetermined by Edman degradation. The sequencing signals obtained wereextremely low as compared with the amount of loaded material (on averagearound 5%). It is therefore likely that the fragments were for the mostpart N-terminally blocked. This supports the idea that proteolysis ofCOL1A1-1 takes place intracellularly. The large amount of COL1A1supplied, allowed nevertheless an easy determination of the N-terminalsequence. The N-terminal sequences obtained are underlined in theprotein sequence (FIG. 8) as encoded by the transfected COL1A1-1 gene.The fragments with an apparent Mw of 38 kD and 18 kD both gave thesequence expected for the N-terminus of the protein, but extended with‘EA’. This extension (or EAEA) is known to be present on some proteinsexpressed from expression vectors utilizing the Saccharomycescerevisiae-derived alpha-mating factor (αMF) prepro signal. This effectis assumed to be due to steric hindrance of STE13 cleavage activity.However, because most of the protein is probably N-terminally blocked,it may well be that this extended version represents only the minorfraction that is sequenceable. Based on the N-terminal and internalsequences, the fragments with an apparent Mw of 38, 24, 18 and 15 kDwere assigned to be fragments consisting of, respectively, residue1-310, 126-310, 1-125, and 42-125 of the target product as shown in FIG.8, and having theoretical Mw's of 28, 16, 12, and 8 kD, respectively.Fragments corresponding to residue 1-41 (theoretical Mw 4 kD) and 42-310(theoretical Mw 24 kD, apparent Mw 34 kD) were not observed. This couldbe due to a (much) more frequent cleavage between residue 125 and 126than between residue 41 and 42.

Cloning and Expression of Mouse COL1A1-2

PCR has been carried out on Mouse 17-day Embryo QUICK-Clone™ cDNA(Clontech) in the same way as mouse COL1A1-1, using C1A1-FW and C1A1-RV2primer. After denaturation at 94° C. for 4 minutes, 35 cycles wereperformed of 1 minute at 94° C., 1 minute at 65° C. and 3 minutes at 72°C. Final extension was at 72° C. for 10 minutes. Agarose gelelectrophoresis shows a 1.8 kb band, which is the size predicted fromthe sequence. The further cloning of COL1A1-2 into the pPIC9 expressionvector has been carried out in the same way as COL1A1-1, yieldingpCOL1A1-2 (FIG. 9).

Pichia pastoris GS115 was transformed with the pCOL1A1-2 vector asdescribed for the pCOL3A1 vector. Sal I-digested DNA was used in orderto specifically generate Mut⁺ transformants. Several transformants wereused for small-scale expression in shaking flasks and one of those wasselected for expression in the fermenter at a 1-100 L scale. FIG. 10(SEQ ID NO:48) shows the expected COL1A1-2 amino acid sequence. Typicalyields are in the range of 4-5 g gelatin/L in the (extracellular)medium, at a dry biomass of 100-120 g/L (about 3 g gelatin/L overall).The target gelatin (FIG. 10) has a theoretical Mw of 53 kD. In agreementwith this value (and with the known anomalous migration of gelatin inSDS-PAGE [10]), a SDS-PAGE band migrating at an apparent Mw of about 74kD was observed (interpolated value obtained with globular protein Mwmarkers). In addition, three shorter products with an apparent Mw of 56,18 and 15 kD were observed (interpolated values). If the proteolyticcleavage would occur at corresponding sites in the COL1A1-1 and COL1A1-2expression products (FIGS. 8, 10), fragments consisting of residue1-595, 126-595, 1-125, and 42-125 of the COL1A1-2 product would beexpected to occur. These would have theoretical Mw's of 53, 42, 12, and8 kD, respectively, corresponding to apparent Mw's of 74, 58, 17 and 11kD. Surprisingly, this corresponds well to the observed apparent Mw's,indicating that cleavage of COL1A1-2 was restricted mainly to the bondbetween residue 125 and 126, and (in addition), the bond between 41 and42. Again, fragments corresponding to residue 1-41 (theoretical Mw 4 kD)and 42-595 (theoretical Mw 50 kD, apparent Mw 70 kD) were not observed.This could be due to a (much) more frequent cleavage between residue 125and 126 than between residue 41 and 42, as mentioned for COL1A1-1.

Cloning and Expression of mouse COL1A1-3

PCR has been carried out on Mouse 17-day Embryo QUICK-Clone™ cDNA(Clontech) in the same way as mouse COL1A1-1, using C1A1-FW and C1A1-RV3primer. After denaturation at 94° C. for 4 minutes, 35 cycles wereperformed of 1 minute at 94° C., 1 minute at 65° C. and 3 minutes at 72°C. Final extension was at 72° C. for 10 minutes. Agarose gelelectrophoresis shows a 2.8 kb band, which is the size predicted fromthe sequence.

The PUC18* plasmid was digested with NcoI and dephosphorylated. TheC1A1-FW/C1A1-RV3 PCR product was digested with NcoI and the resulting2.5 kb fragment was gel purified and ligated into the NcoI digested anddephosphorylated vector. After transformation of E. coli XL1Blue,correct orientation of the insert in the resulting clones was verifiedby PvuII digestion. The further cloning of COL1A1-3 into the pPIC9expression vector has been carried out in the same way as described forCOL1A1-1, yielding pCOL1A1-3 (FIG. 11).

Pichia pastoris GS115 was transformed with the pCOL1A1-3 vector asdescribed for the pCOL3A1 vector. Sal I-digested DNA was used in orderto specifically generate Mut⁺ transformants. Several transformants wereused for small-scale expression in shaking flasks and one of those wasselected for expression in the fermenter at a 1-100 L scale. FIG. 12(SEQ ID NO:49) shows the expected COL1A1-3 amino acid sequence. Typicalyields are in the range of 4-5 g gelatin/L in the extracellularcompartment (determined after acetone fractionation, as describedbelow), at a dry biomass of 100-120 g/L (about 3 g gelatin/L overall).The target gelatin (FIG. 12) has a theoretical Mw of 72 kD. In agreementwith this value, an SDS-PAGE band migrating at an apparent Mw of about100 kD was observed (interpolated value obtained with globular proteinMw markers). In addition, three shorter products with an apparent Mw of85, 18 and 15 kD were observed (interpolated values). If the proteolyticcleavage would occur at homologous sites in the COL1A1-1 and COL1A1-3expression products (FIGS. 8, 12), fragments consisting of residue1-812, 126-812, 1-125, and 42-125 of the COL1A1-3 product would beexpected to occur. These would have theoretical Mw's of 72, 60, 12, and8 kD, respectively, corresponding to apparent Mw's of 100, 84, 17 and 11kD. Surprisingly, this corresponds well to the observed apparent Mw's,indicating that cleavage of COL1A1-3 was restricted mainly to the bondbetween residue 125 and 126, and (to a lesser extent), the bond between41 and 42. Again, fragments corresponding to residue 1-41 (theoreticalMw 4 kD) and 42-812 (theoretical Mw 68 kD, apparent Mw 96 kD) were notobserved. This could be due to a (much) more frequent cleavage betweenresidue 125 and 126 than between residue 41 and 42, as mentioned forCOL1A1-1.

Table 1 summarizes the apparent COL1A1 fragment sizes derived bycomparison with molecular weight marker proteins (LMW Calibration kit;Pharmacia), together with the size calculated from the sequence.

TABLE 1 Apparent and theoretical molecular weights of COL1A1 fragmentsApparent Molecular molecular weight Molecular weight on corrected forweight SDS-PAGE 40% slower calculated from gel migration Residue thesequence Gene product 100 kD  71 kD 1-812 72 kD (1A1-3) 85 kD 61 kD125-812  61 kD (1A1-3) 74 kD 53 kD 1-595 54 kD (1A1-2) 56 kD 40 kD125-595  42 kD (1A1-2) 38 kD 27 kD 1-310 28 kD (1A1-1) 24 kD 17 kD125-310  16 kD (1A1-1) 18 kD 13 kD 1-125 12 kD (1A1-1,2,3) 15 kD 11 kD42-125   8 kD (1A1-1,2,3)

It is clear from table 1 that the ‘MGPR’ model fits the actual foundfragment sizes well.

One would also expect fragments of residue 1-41 (4 kD; 1A1-1,2,3) and42-310 (theoretical Mw: 24 kD apparent Mw: 34 kD; 1A1-1), 42-595(theoretical Mw: 50 kD apparent Mw: 70 kD; 1A1-2), or, 42-595(theoretical Mw: 68 kD apparent Mw: 96 kD; 1A1-3). The fact that thesefragments are not seen on gel, may be explained by assuming that therate of cleavage at the second ‘MGPR’ site is much higher than that atthe first site. This means that if the protein molecule is cut, it willalways first occur at the second site. This difference in cleavage ratesmay be explained by the fact that the first site is preceded by aproline residue which may sterically hinder the protease.

Mouse COL1A1-1, COL1A1-2 AND COL1A1-3 ‘RGPM’ (SEQ ID NO:16) Mutants

We considered the possibility that the same amino acid sequence,functioning as recognition site for proteolytic enzyme(s), would beresponsible for the degradation of all COL1A1 products (COL1A1-1,COL1A1-2, COL1A1-3). Surprisingly, both internal N-terminal sequencesobtained from the 15 kD and 24 kD COL1A1-1 fragments were preceded bythe same sequence ‘MGPR’ (SEQ ID NO:14). Moreover, this sequence occursonly twice in the mouse COL1A1-1, COL1A1-2, or COL1A1-3 genes, viz. atresidue 83-41 and 122-125. (As compared to COL1A1-1, COL1A1-2 andCOL1A1-3 do not contain additional MGPR-sites). Also, the COL3A1fragment from the rat did not contain such a site. This correspondsnicely to the observed cleavage pattern. Therefore, we think that ‘MGPR’(SEQ ID NO:14) is a motif recognized by a specific protease, resultingin the cleavage of the COL1A1 proteins. This MGPR (SEQ ID NO:14)protease recognition site has not been described previously. A moregeneralized representation of the motif could possibly be MXXR, MXX[RK],or even [MLIV]XX[RKH] (SEQ ID NO:17). The former motifs are indeedpresent only twice in the entire mouse COL1A1 gene and are absent in theCOL3A1 fragment, while the latter motif is so broad that it includesnon-cleaved sites: MKGH (SEQ ID NO:18) at residue 85-88 and VGAK (SEQ IDNO:19) at residue 169-172 in COL1A1, and IKGH (SEQ ID NO:20) at residue198-201 in COL3A1. Thus, MXXR or MXX[RK] are more likely generalizedmotifs than [MLIV]XX[RKH (SEQ ID NO:17). FIG. 13 (SEQ ID NO:50) showsthe ‘MGPR’ (SEQ ID NO:14) motifs in the COL1A1-2 sequence. It is to beexpected that this proteolytic cleavage site is only recognized by theenzymes involved if it occurs in relatively open, unfolded structures(like in our gelatins), but not so easily in more compactly foldedstructures (like in globular proteins). Thus, it may be important onlyin certain classes of proteins and polypeptides, including gelatins andunfolded collagens.

In order to be able to produce full-length COL1A1-1, COL1A1-2 ANDCOL1A1-3 without the occurrence of the three other main bands, the‘MGPR’ (SEQ ID NO:14) motifs should be removed by site-directedmutagenesis. In order to maintain the original amino acid composition ofnatural COL1A1 gelatin, the ‘MGPR’ (SEQ ID NO:14) motif was removed byconverting it to ‘RGPM’ (SEQ ID NO:16). Two pairs of complementaryprimers were synthesized:

COL1A1MUT1FW: (SEQ ID NO: 16) R G P M (SEQ ID NO: 21)5′-GAG-CCT-GGC-GGT-TCA-GGT-CCA-CGA-GGT-CCA-ATG- GGT-CCC-CCT-GG-3′COL1A1MUT1RV: (SEQ ID NO: 22)5′-CC-AGG-GGG-ACC-CAT-TGG-ACC-TCG-TGG-ACC-TGA- ACC-GCC-AGG-CTC-3′COL1A1MUT2FW: (SEQ ID NO: 16) R G P M (SEQ ID NO: 23)5′-GGA-GCT-CCT-GGC-CAG-CGA-GGT-CCA-ATG-GGT-CTG- CCC-GGT-GAG-AG-3′COL1A1MUT2RV: (SEQ ID NO: 24)5′-CT-CTC-ACC-GGG-CAG-ACC-CAT-TGG-ACC-TCG-CTG- GCC-AGG-AGC-TCC-3′ Note:mutant positions are underlined; the original C of the Pro residue hasbeen converted into A to avoid the generation of an NcoI site. Threeprimer combinations were used: 1. 5′AOX1 primer and COL1A1MUT1RV 2.COL1A1MUT1FW and COL1A1MUT2RV 3. COL1A1MUT2FW and 3′AOX1

The reactions contained: 1.25 U Pwo polymerase (Eurogentec), 50 pmol ofeach primer, 0.2 mM dNTPs (Pharmacia), 1× Pwo buffer (Eurogentec) and 15ng pCOL1A1-1 template DNA in a total reaction volume of 50 μl. ThePCR-machine used was the GeneAmp 9700 (Perkin-Elmer). After an initialincubation at 94° C. for 5 minutes, 18 cycles were performed consistingof 30 seconds at 94° C., 30 seconds at 60° C. and 45 seconds at 72° C.Final extension was performed at 72° C. for 10 minutes. Agaroseelectrophoresis of the PCR-reactions revealed products of the expectedsizes (0.5, 0.3 and 0.7 kb respectively). The bands were cut out fromthe gel and purified. The isolated fragments were then subjected tooverlap-extension PCR. Approximately 0.1 pmol of each fragment was mixedtogether. 50 pmol of 5′AOX1 and 3′AOX1 primer were added, as well as Pwopolymerize, dNTPs and buffer as described above. Cycling conditions werethe same as described above with the exception that extension at 72° C.was performed for 90 seconds instead of 45 seconds. Agarose gelelectrophoresis revealed the expected 1.5 kb product. The remainder ofthe PCR reaction was purified using the QIAquick PCR Purification Kit(Qiagen). The purified DNA was then digest-d with BamHI/ApaI, afterwhich the resulting 1.0 b fragment was purified from gel. E. coli strainJM110 was transformed with pCOL1A1-1, pCOL1A1-2 and pCOL1A1-3, in orderto remove the dcm methylation of the ApaI site. After DNA isolation, theplasmids were digested with BamHI/ApaI. The resulting vector fragmentsof 7.9, 8.3 and 9.5 kb, respectively, were purified from agarose gel andligated to the BamHI/ApaI digested PCR-product. E. coli XL1-Blue wastransformed with these ligation reactions and plasmid DNA ofPCR-verified insert containing clones was isolated and verified byautomated sequencing. The mutant plasmids pCOL1A1-1*, pCOL1A1-2* andpCOL1A1-3* thus created were digested with SalI and used to transformPichia pastoris strain GS115. Small- and fermentor-scale expression wasperformed as described for COL1A1-1.

The SDS-PAGE analysis clearly shows that only one major band of theexpected full-length size is formed, for COL1A1-1* as well as COL1A1-2*and COL1A1-3*.

Construction and Expression of Synthetic Gene.

A synthetic gene, encoding a polar gelatin (P monomer) was constructed boverlap extension PCR. The theoretical molecular weight and isoelectricpoint are 9.1 kD and 4.9, respectively. The gene was designed to havethe codon usage of Pichia pastoris highly expressed genes (Sreekrishna.K. and Kropp, K. E. (1996) Pichia pastoris, Wolf, K. (Ed),Nonconventional yeasts in biotechnology. A handbook. Springer-Verlag,pp. 62 203-6'253). Two separate PCR reactions were performed, using thefollowing oligonucleotides:

1 pmol OVL-PA-FW, 1 pmol OVL-PA-RV, 50 pmols HLP-PA-FW and 50 pmolsHLP-PA-RV. 1 pmol OVL-PB-FW, 1 pmol OVL-PB-RV, 50 pmols HLP-PB-FW and 50pmols HLP-PB-RV.

Oligonucleotide sequences were as follows:

HLP-PA-FW: (SEQ ID NO: 25) 5′-GCGCTCGAGAAAAGAGAGGCTGAAGC-3′ OVL-PA-FW:(SEQ ID NQ: 26) 5′-GCGCTCGAGAAAAGAGAGGCTGAAGCTGGTCCACCCGGTGAGCCAGGTAACCCAGGATCTCCTGGTAACCAAGGACAGCCCGGTAACAAGGGTTCTC CAGGTAATCCA-3′OVL-PA-RV: (SEQ ID NO: 27)5′-TGAGAACCTTGTGGACCGTTGGAACCTGGCTCACCAGGTTGTCCGTTCTGACCAGGTTGACCAGGTTGACCTTCGTTTCCTGGTTGACCTGGATT ACCTGGAGAACCCTT-3′HLP-PA-RV: (SEQ ID NO: 28) 5′-TGAGAACCTTGTGGACCGTTGGAA-3′ HLP-PB-FW:(SEQ ID NO: 29) 5′-TTCCAACGGTCCACAAGGTTCTCA-3′ OVL-PB-FW: (SEQ ID NO:30) 5′-TTCCAACGGTCCACAAGGTTCTCAGGGTAACCCTGGAAAGAATGGTCAACCTGGATCCCCAGGTTCACAAGGCTCTCCAGGTAACCAAGGTTCCCCT GGTCAGCCAGGTAACCCT-3′OVL-PB-RV: (SEQ ID NO: 31)5′-GCGTCTGCAGTACGAATTCTATTAGCCACCGGCTGGACCCTGGTTTCCTGGTTTACCTTGTTCACCTGGTTGACCAGGGTTACCTGGCTGACCAG GGGAACCTTGGTT-3′HLP-PB-RV: (SEQ ID NQ: 32) 5′-GCGTCTGCAGTACGAATCTATTAGC-3′

The 50 μl PCR reactions contained 0.2 mM dNTP's (Pharmacia), 1× Pwobuffer (Eurogentec) and 1.25 U Pwo polymerase (Eurogentec). Reaction 1involved 18 cycles consisting of 15 seconds at 94° C. and 15 seconds at72° C. Reaction 2 involved a touchdown PCR, whereby each cycle consistedof 15 seconds at 94° C., 15 seconds at the annealing temperature and 15seconds at 72° C. The annealing temperature was lowered from 72° C. to68° C. in the first 5 cycles, after which 20 additional cycles at anannealing temperature of 67° C. were performed.

The PCR products were isolated from agarose gel. 0.3 pmols of eachfragment and 50 pmols of the outer primers HLP-PA-FW and HLP-PB-RV weresubjected to overlap extension PCR. 25 cycles consisting of 15 secondsat 94° C., 15 seconds at 67° C. and 15 seconds at 72° C. were performed.The resulting 0.3 kb PCR fragment was digested with XhoI/EcoRI andcloned in cloning vector pMTL23. The sequence of the gene was verifiedby automated DNA sequencing.

In order to create a P tetramer (P4; theoretical molecular weight 36.8kD), the fragment was released by digesting the vector withDraIII/Van91I. In a separate reaction the vector was digested withVan91I and dephosphorylated. The DraIII/Van91I fragment was theninserted into this Van91I digested vector. This yielded a vectorcontaining a P dimer. This dimer was released by digestion withDraIII/Van91I and reinserted into the Van91I site of the dimer bearingvector, yielding the P tetramer (P4). The P and P4 fragments were thencloned into the XhoI/EcoRI sites of vector pPIC9. The encoded amino acidsequence of the mature (processed) P monomer and tetramer are asfollows:

Monomer (P (SEQ ID NO: 33)):   1 GPPGEPGNPG SPGNQGQPGN KGSPGNPGQPGNEGQPGQPG QNGQPGEPGS NGPQGSQGNP  61 GKNGQPGSPG SQGSPGNQGS PGQPGNPGQPGEQGKPGNQG PAGG Tetramer (P4 (SEQ ID NO: 34)):   1 GPPGEPGNPG SPGNQGQPGNKGSPGNPGQP GNEGQPGQPG QNGQPGEPGS NGPQGSQGNP  61 GKNGQPGSPG SQGSPGNQGSPGQPGNPGQP GEQGKPGNQG PAGEPGNPGS PGNQGQPGNK 121 GSPGNPGQPG NEGQPGQPGQNGQPGEPGSN GPQGSQGNPG KNGQPGSPGS QGSPGNQGSP 181 GQPGNPGQPG EQGKPGNQGPAGEPGNPGSP GNQGQPGNKG SPGNPGQPGN EGQPGQPGQN 241 GQPGEPGSNG PQGSQGNPGKNGQPGSPGSQ GSPGNQGSPG QPGNPGQPGE QGKPGNQGPA 301 GEPGNPGSPG NQGQPGNKGSPGNPGQPGNE GQPGQPGQNG QPGEPGSNGP QGSQGNPGKN 361 GQPGSPGSQG SPGNQGSPGQPGNPGQPGEQ GKPGNQGPAG G

In order to prevent possible C-terminal degradation of the P and P4gelatins, constructs were created that have identical sequences as P andP4, but which have a C-terminal Pro instead of a Gly residue (PC andP4C, respectively).

The vectors were linearized by digestion with SalI and were used totransform Pichia pastoris strain GS115. Fermentations were performed asdescribed for COL1A1 (i.e. growth at pH 3 and in the presence ofcasamino acids). Culture supernatants were analyzed by SDS-PAGE andrevealed protein bands having the expected N-terminal amino acidsequences. The yield was calculated to be 1 g-ram/liter fermentationmedium. The products could be purified by acetone fractionation asdescribed for the native gelatins (i.e. removal of endogenous proteinsat 40% acetone and precipitation of gelatin at 80°, acetone).

Expression/Production of Gelatin in a Fermentor

Fed-batch fermentations were performed according to the Pichiafermentation process guidelines of Invitrogen. Cells were grown in aI-liter fermentor (Applikon) in the initial experimental stages tooptimize protein production. Thereafter cells were grown in a 20-literor a 140-liter fermentor (Biobench 20, Bio-pilot 140, Applikon) forpilot scale production of collagen. Working volumes were I-liter,15-liter and 100-liter, respectively. AD1020 controllers (Applikon) wereused to monitor and control the fermentation parameters. The programBioXpert (Applikon) was used for data storage. Dissolved oxygen levelswere monitored in the fermentor using an oxygen electrode (Ingold for1-liter fermentations. Mettler Toledo for larger scale fermentations).Agitation (500-1000 rpm) and aeration (1-2 vvm, i.e. 1-2 LL⁻¹ min⁻¹)were manually adjusted to keep the dissolved oxygen concentration above20%, pH was measured by a pH electrode (Broadly James cooperation) andautomatically kept at pH 3.0 or pH 5.0 by addition of ammonium hydroxide(25%), which also served as nitrogen source for growth of themicroorganisms. An anti foam-electrode was used to prevent excessivefoaming. When necessary, the anti foam Structol J673 (Schill andSeilacher, Hamburg, Germany) was used. Growth of the microorganisms wasmonitored by determination of the cell dry weight. A calibration curvewas made by which cell wet weight could be converted into cell dryweight. Cell wet weight was determined after centrifugation of 2ml-samples for 5 min at 15.000 rpm and removing the supernatant. Celldry weight was determined after addition of 200 μl of cells to apre-dried filter (0.45 μm membrane, Schleichner & Schüll, Dassel,Germany), washing with 25 ml of deionized water and drying e.g. in amicrowave oven for 15 minutes at 1000 W. Cell dry weight wasapproximately a factor 3 lower than call wet weight. Precultures werestarted from colonies on a MGY plate, in flasks containing a total of10% of the initial fermentation volume of MGY. The volume of the mediumwas ≦20% of the total flask volume. Cells were grown at 30° C. at 200rpm in a rotary shaker (Gallenkamp) for 24-60 hours.

Fermentation Medium

The fermentation basal salts medium in the fermentor contained perliter: 26.7 ml of phosphoric acid (85%), 0.93 g calcium sulfate, 18.2 gpotassium sulfate. 14.9 g magnesium sulfate. 7H₂O, 4.13 g potassiumhydroxide and 40.0 g glycerol. An amount of 4.3 ml of PTM₁ trace saltswas added per liver of fermentation basal salts medium. PTM₁ trace saltscontained per liter: 4.5 g cupric chloride.2H₂O. 0.09 g potassiumiodide, 3.5 g manganese chloride.4H₂O, 0.2 g sodium molybdate.2H₂O. 0.02g boric acid, 1.08 g cobalt sulfate.7H₂O. 42.3 g zinc sulfate.7H₂O, 65.0g ferrous sulfate. 7HO. 0.2 g biotin and 5.0 ml sulfuric acid. Tracesalts were filter sterilized (pore size 0.22 μm, Costar, USA). Casaminoac ds (caseine hydrolysate Merck) were separately sterilized and addedto the fermentation medium in an amount or 5 g/l when collagen type Ifrom the mouse was expressed (COL1A1-1, COL1A1-1*, COL1A1-2. COL1A1-2*,COL1A1-3, COL1A1-3*). During the fermentation after 50 hours a furtheramount of 5 g/l of sterile casamino acids was added to the fermentationmedium.

Fermentation of Mut⁻ Cultures

The fermentor was sterilized with the fermentation basal salts medium.The 20-liter and 120-liter fermentor were sterilized in situ withinitial medium volumes of 5-7.5 l and 50-liter, respectively. The1-liter fermentor was sterilized with 500 ml medium in an autoclave.After sterilization the temperature was set at 30° C., agitation andaeration were set at 500 rpm and 1 vvm (i.e. I LL⁻¹ min⁻¹),respectively. The pH was adjusted to set point (usually pH 5.0) with 25%ammonium hydroxyde. Trace salts were aseptically added to the medium.The fermentor was inoculated with 10% of the initial fermentation volumeof precultured cells in MGY. The batch culture was growl until theglycerol was completely consumed (18-24 hours). This was indicated by anincrease of the dissolved oxygen concentration to 100%. Cell dry weightwas 25-35 g/l in this stage. Thereafter the glycerol fed-batch phase wasstarted by initiating a 50% (v/v) glycerol feed containing 12 ml PTM₁trace salts per liter of glycerol. The glycerol feed was set at 13ml/h/liter initial fermentation volume. The glycerol feed was carriedout for 4 hours, or overnight in the case of a long lag phase. Duringthe glycerol batch phase the pH of the fermentation medium was loweredto 3.0. The protein induction phase was initiated by starting a 100%methanol feed containing 12 ml PTM₁ trace salts per liter of methanol.The feed rate was set to 3 ml/h/liter initial fermentor volume. Duringthe first hours methanol accumulated in the fermentor. After 2-4 hoursdissolved oxygen levels decreased due to adaptation to methanol. Themethanol feed was increased to 6 ml/h/initial fermentor volume in thecase of a fast dissolved oxygen spike. If the carbon source is limiting,shutting of the carbon source causes the culture to decrease itsmetabolic rate and the dissolved oxygen concentration rises (spike).After an additional 2 hours the methanol rate was increased to 9ml/h/liter, initial fermentor volume. This feed rate was maintainedthroughout the remainder of the fermentation. The fermentation wasstopped after 70-130 h methanol fed-batch phase. During the fermentationsamples were taken of 2 ml, centrifuged (5 min, 15.000 rpm) and thesupernatant was stored at −20° C.

Concentration of gelatin and total protein was determined afterfiltration of the samples (0.22 μm) and subsequent acetone fractionation(40 vol-%, followed by 60-80 vol-% acetone). The BCA protein assay(Pierce) was routinely used, with gelatin from Merck as a reference.According to SDS-PAGE and analysis of the amino acid composition, thenon-collagenous proteins precipitated at 40% acetone, while the COL3A1and COL1A1 fragments precipitated at 60-80%. At 60% acetone,preferentially the higher molecular weight gelatin componentsprecipitated. At increasing acetone concentration, increasingprecipitation was obtained for the main degradation products describedabove. At 80%, all of the main degradation products were recovered inthe precipitate (as checked with SDS-PAGE). Small peptides and other lowmolecular weight contaminants remained in solution at 80% acetone.

At the end of the fermentation, the cells were removed by centrifugation(10.000 rpm. 30 min. 4° C.) in the case of the 1-liter fermentation.Cells were removed by micro filtration in the case of the 20-literfermentation. The cell broth was first applied to a micro filtrationmodule containing a polyether sulfone membrane with 0.50 μm pore size(type MF 05 M2 from X-Flow, fitted in a RX 300 filtration-module fromX-Flow). Thereafter the supernatant was applied to a similar type ofmicro filtration module containing a polyether sulfone membrane with 0.2μm pore size (type MF 02 M1, similarly from X-Flow). In the case of the120-liter fermentation cells were removed by a pilot plant scale microfiltration unit containing a polyether sulfone membrane with 0.2 μm poresize (type MF 02 Ml, from X-Flow, fitted into a R-10 membrane module).These filtration units are mentioned merely as examples. It will beunderstood that any suitable micro filtration system could be applied toremove the cells. Optionally, the bulk of cells and debris was removedby centrifugation, and only the supernatant and the medium used to washthe cells was applied to the microfiltration units. Alternatively, it ispossible to recover the product from the fermentation broth and separateit from the cells by directly applying the fermentation broth to asuitable expanded bed chromatography system, using a resin thatspecifically binds the gelatin produced. We successfully used SPSepharose XL Streamline from Pharmacia as a cation exchanger in expandedbed mode, at pH 3-4.

Fermentation of Mut^(s) Cultures

Glycerol batch and fed-batch phase were performed as described for themut⁻ cultures. Since Mut^(s) cultures metabolize methanol poorly, theiroxygen consumption is very low. Therefore spikes of the dissolved oxygenconcentration cannot be used to evaluate the culture. The methanol feedwas adjusted to maintain an excess of methanol in the medium which doesnot exceed 0.3%. The methanol feed was initiated at 1 ml/h/liter initialfermentor volume and increased slowly to 3 ml/h liter. The totalfermentation time required when using Mut^(s) cultures was comparativelylonger than when Mut⁻ cultures were used.

Preparative Purification of Collagen/Gelatin on a Preparative Scale

After the micro filtration step, two alternative purification strategieswere followed (see I, II below).

I. Purification by Differential Precipitation Acetone Fractionation

Collagen type I and type III were partly purified from batches of 500 mlto 2 liter of supernatant by a 40-80% acetone fractionation. At 40%acetone, the non-collagenous proteins (from Pichia) were precipitated,while at 60-80% acetone, collagen as well as collagen breakdown productswere precipitated, as shown by SDS-PAGE and analysis of the amino acidcomposition. At 60% acetone, preferentially the higher molecular weightgelatin components precipitated. At increasing acetone concentration,increasing precipitation was obtained for the main degradation productsdescribed above. At 80% all of the main degradation products wererecovered in the precipitate (as checked with SDS-PAGE). Small peptidesand other low molecular weight contaminants remained in solution at 80%acetone. Acetone was cooled for 2-4 hours at −20° C. An amount of 40% ofice-cold acetone (v/v) was added slowly to the pre-cooled supernatantfrom the fermentation at 4° C. under magnetic stirring. Supernatant wasstirred overnight at 4° C. Precipitated proteins and particles wereremoved by centrifugation (4° C., 10.000 rpm, 30 min). The pellet wasresuspended in 40% ice-cold acetone and again centrifuged. Both 40%acetone supernatant fractions were pooled. Thereafter the supernatantwas brought to 60-80% acetone (v/v) and stirred overnight. Precipitatedproteins were collected by centrifugation. The pellet was resuspended in60-80% acetone and centrifuged again. The pellet was dissolved in anappropriate amount of a 5 ml acetic acid; ammonium hydroxide buffer atpH 3.0 (buffer A) to a protein concentration of 20-50 gl.

Ammonium Sulphate Precipitation

Polysaccharides were subsequently removed by precipitation of thegelatin/collagen at 60% saturation of ammonium sulphate, where thepolysaccharides remained in solution. Ammonium sulphate was slowly addedto 60% saturation at 4° C. After 60 min stirring the sample wascentrifuged (30 min, 4° C., 10.000 rpm). The pellet was resuspended in60% ammonium sulphate and again centrifuged. If more than 1% (w/w)polysaccharides or sugars remained present, the complete ammoniumsulphate precipitation procedure described above was repeated aftercomplete redissolution of the gelatin/collagen in the absence ofammonium sulphate. Finally, the pellet was dissolved in deionized wateror in buffer A to a protein concentration of 20-50 g/l. The pH of thesample was adjusted to 3.0. The sample was desalted by dialysis againstbuffer A, which was refreshed every 4 hours. Dialysis membranes ofregenerated cellulose (Spectra Por®, from Spektrum) were used with amolecular weight cut-off of 8 kD. The dialysis was stopped after 2-7days at the moment that the conductivity of the sample was judged to besufficiently low (topically 20-150 μS.cm⁻¹ above background).Conductivity was measured with a digital conductivity meter(Radiometer), calibrated with 1 mM and 10 mM KCl solutions (140 and 1400μS.cm⁻¹, respectively). As an alternative to dialysis, ultrafiltrationand diafiltration were used to desalt the samples and (optionally) toconcentrate them. Where applicable, the product was subsequentlypre-dried (optional) by precipitation with high concentrations ofacetone and evaporation of the acetone, and finally freeze-dried.

II. Purification by Cation-Exchange Chromatography

The cation-exchange resin was SP Sepharose XL (Pharmacia Biotech), butother suitable resins could also be used. The purification was carriedout at several scales. Thus, 25 mL bed in a XK16 column (Pharmacia) wasused. Runs were performed with a FPLC (Pharmacia). Bed height was 12.5cm. Flow rates were typically 1 mL/min. At an intermediate scale, a 100mL bed was used. Runs being controlled by an Äkta Explorer integratedpump/processor/multiple valve/multiple detector unit (Pharmacia). Onpilot scale a 2 liter bed in an Index 140 200 column was used. Bedheight was at least 13 cm. Runs were performed with the ÄKTA explorerpump-processor unit (Pharmacia) or other pump systems. Flow rates were50-100 ml/min, or higher. As an example, the following buffer system andelution conditions were used. Buffer X was a 5 mM citric acid buffer atpH 3.2, buffer Y a 5 mM citric acid buffer with 1 NM NaCl at pH 3.0. Thecolumn was equilibrated with 2-5 bed volumes of buffer X. The protein ofinterest was eluted with a linear gradient of 0-0.5 M NaCl in 5-10column volumes. The main band of collagen type III eluted at 50-100 mMNaCl. The main band of collagen type I eluted at 70 mM NaCl, the otherbands between 30-150 mM NaCl, in agreement with their theoreticalisoelectric points. The column was cleaned with 1 bed volume of bufferY. On a pilot scale the pooled fractions were desalted and concentrated,e.g. by addition of 80% acetone, and subsequently freeze-dried.

Characterization of the Gelatin/Collagen Product

The amino acid composition was determined after complete HCl-mediatedhydrolysis of the peptide bonds at very low pH and elevated temperature,followed by derivatisation of the amino acids with a fluorophore, andHPLC.

The percentage Gly expected from pure collagen is 33%. This offers ameans of estimating the purity of produced recombinant gelatins. Inorder to correct for the percentage of Gly in endogenously secretedproteins of Pichia pastoris, amino acid composition analysis wasperformed on fermentation supernatant of a Mut⁻ transformant of pPIC9.The percentage Gly found was 9%. The purity of a sample can now beestimated by the formula:

(% Gly-9)/(33−9)=(%/Gly-9)24.

After dissolution of samples in MilliQ water, the following assays wereperformed.

The protein content was determined by the BCA assay from Pierce, usinggelatin from Merck as a reference.

The protein Mw distribution was determined by SDS-PAGE.

The sugar content was determined by a phenol-based assay. 200 μL sampleswere mixed with 200 μL 5% (w/w) phenol. After thorough mixing using aVortex mixer, 1 mL of concentrated sulphuric acid was added. Aftermixing, the samples were incubated for 10 min at room temperature and,subsequently, for 20 min. at 30° C. After cooling, the light absorptionof the samples at 485 nm was determined. Starch, glucose and sucrosewere used to prepare calibration curves.

The DNA content was determined by mixing aliquots of diluted SYBR® GreenI nucleic acid “gel” stain (10.000× conc. In DMSO) from Molecular Probeswith our samples. After thorough spectral analysis, the excitationwavelength was chosen to be 490 nm, and the emission wavelength 523 nm.The calibration was by subsequent addition of known amounts of DNA tothis same mixture, as internal standards. Thus, a calibration curve wasconstructed. Furthermore, it was checked that subsequent addition ofDNA-degradinq enzyme resulted in complete break down of the fluorescentsignal.

A quantitative indication of the RNA plus DNA-content was subsequentlyobtained by using SYBR® Green II “RNA gel stain”, instead of SYBR® GreenI. After thorough spectral analysis, the excitation wavelength waschosen to be 490 nm, and the emission wavelength 514 nm. Calibration wasby subsequent addition of known amount of RNA. The resulting value wasdenounced the “RNA” content of the sample. In the absence of DNA, itcorresponded to the true RASA content. When present, the DNA-associatedfluorescence may have biased the RNA values, although a final additionof RNAse was used to discern the DNA- and RNA-derived contributions tothe fluorescence.

The conductivity of the samples was measured with a digital Radiometerconductivity meter, checking that 1 and 10 KCL solutions in MilliQ watergave readings of 140 and 1400 μS.cm⁻¹, respectively.

Data on Purity of Some Gelatin Batches Produced in Accordance with theInvention (Examples)GATO4a (col3a1)

-   -   about 2.4 gram    -   purification:    -   micro filtration, precipitation (1× acetone fractionation        (40%/80%), 1× (NH₄)₂SO₄), dialysis against 5 mM        CH₃COOH/CH₃COO⁻-buffer keeping the sample below pH 4 (initial pH        about 3.5; buffer prepared by dilution from 500 mM acetic acid        adjusted to pH 3.0 with 25% NH₄OH), lyophilization    -   DNA: <1 ppm (w/w)    -   RNA: 12.7 ppm (w/w)    -   total sugars: 4.5% (w/w)    -   gelatin was not degraded during purification (SDS-PAGE: FIG. 14)        GATO4b (col3a1, Further Purified than GATO4a)    -   about 1 gram    -   purification:        -   further purified from GATO4a by repeated (2× additional)            ammonium sulphate precipitation followed by dialysis against            5 mM CH₃COOH/CH₃COO⁻-buffer keeping the sample belong pH 4            (initial pH about 3.5; buffer prepared by dilution from 500            mM acetic acid adjusted to pH 3.0 with 25% NH₄O—H),            lyophilization    -   DNA: 0.56 ppm (w/w)    -   RNA: 3.2 ppm (w/w)    -   total sugars: 0.94% (w/w)    -   gelatin was not degraded during purification.    -   specific conductance after dialysis about 180 μScm⁻¹ at 10 g        gelatin/L (specific conductance of buffer about 100 μScm⁻¹)    -   specific conductance after lyophilization and dissolving a        sample: 180 μScm⁻¹ at 10 g gelatin/L, 100 μScm⁻¹ at 5 g        gelatin/L        GATO5 (col1a1-1)    -   about 0.9 gram    -   purification:        -   micro filtration, precipitation (1× acetone fractionation            (40%/80/80), 1× (NH₄)₂SO₄), dialysis against 5 mM            CH₃COOH/CH₃COO⁻-buffer keeping the sample below pH 4            (initial pH about 3.5; buffer prepared by dilution from 500            mM acetic acid adjusted to pH 3.0 with 25% NH₄OH),            lyophilization    -   DNA: <1 ppm (w/w)    -   RNA: 87 ppm (w/w)    -   total sugars: 4.5% (w/w)    -   gelatin was not degraded during purification (SDS-PAGE: FIG. 15)        GATO6 (col3a1 Purified by Expanded Bed Cation Exchange        Chromatography)    -   about 50 mg    -   purification:        -   expanded bed cation exchange chromatography at pH 3-3.5            (SP-Sepharose-XL “Streamline” resin from Pharmacia Biotech;            sugar content after sub-optimal washing of the column and            elution at 0.3 M NaCl: 1.8% (w/w)), further removal of sugar            by a single (NH₄)₂SO₁-precipitation, followed by dialysis            against 5 mM NH₄ ⁻/CH₃COO⁻-buffer (pH about 3.5),            lyophilization    -   DNA: <1 ppm (w/w, already before (NH₄)₂SO₄-precipitation and        dialysis)    -   RNA: <9 ppm (w/w, already before (NH₄)₂SO₄-precipitation and        dialysis)    -   total sugars: 1.1%    -   specific conductance after dialysis about 94 μScm⁻¹ at 0.55 g        gelatin/L (specific conductance of buffer: about 100 μScm¹).    -   gelatin was not degraded during purification        GATO7 (col1a1-2)    -   400 mg    -   purification:        -   micro filtration, precipitation (1× acetone fractionation            (40%/71.5%), 3× (NH₄)₂SO₄), pre-desalting by acetone            precipitations: 1×71.5%, 1×80%, dialysis against MilliQ            water, lyophilization    -   DNA: 0.79 ppm    -   RNA: 9.5 ppm    -   total sugars: 0.7% (w/w)    -   specific conductance after dialysis about 15.5 μScm⁻¹ at 4 g        gelatin/L    -   gelatin was not degraded during purification        GATO8 (col3a1)    -   about 6 g    -   purification:        -   microfiltration, dilution, cation exchange chromatography in            a 2.1 litre bed SP Sepharose-XL from Pharmacia Biotech            equilibrated with 20 mM citrate, pH 3.5 and elution at 0.15            M NaCl in the same buffer over a gradient of 0-1 M NaCl,            concentration, partial desalination with 80% acetone,            centrifugation, resolubilisation in MilliQ water, dialysis            against MilliQ water and lyophilisation    -   DNA: 1.55 ppm (w/w)    -   RNA: 10.9 ppm (w/w)    -   total sugars: 1.2% (w/w)    -   specific conductance after dialysis about 90 μScm⁻¹ at 7.5 g        gelatin/L    -   gelatin was not degraded during purification

FIG. 16 shows the result of purification.

GATO9 (col1a1-1)

-   -   1.7 g    -   purification: see GATO8 with one difference i.e. elution from        the cationic exchanger in a 1 salt step at 0.75 M NaCl.    -   DNA: <1 ppm (w/w)    -   RNA: 1.3 ppm (w/w)    -   total sugars: 2.2% (w/w)    -   specific conductance after dialysis about 70 μScm⁻¹ at 12 g        gelatin/L    -   gelatin was not degraded during purification        GATO10 (col1a1-2) Wherein Both MGPR Sequences Have Been Changed        to RGPM    -   6 g    -   purification: see GATO8    -   DNA: 0.04 ppm (w/w)    -   RNA: 2 ppm (w/w)    -   total sugars: 2% (w/w)    -   gelatin was not degraded during purification    -   N terminal amino acid sequence was as expected with Glu-Ala as N        terminal extension due to incomplete removal of the propeptide.

The results are shown in FIG. 18.

In conclusion the amino acid composition of all samples matched thetheoretical composition. The contamination with foreign protein was verylow. On a glycine basis GATO4-GATO8 have less than 1% foreign protein ascontaminant. GATO9 and GATO10 have less than 5%.

DESCRIPTION TO THE FIGURES

FIG. 1: vector pCOL3A1

FIG. 2: PCR primers for pCOL3A1 construct control (5′AOX1—SEQ ID NO:35;3′AOX1—SEQ ID NO:36; αMF—SEQ ID NO:37)

FIG. 3: The expected COL3A1 sequence (SEQ ID NO:38). The N terminal Y isderived from the pPIC 9 vector. The rest of the sequence is derived fromCOL3A1 of the rat. The underlined sequences correspond to the N terminalsequences obtained for COL3A1 fragments.

FIG. 4: vector pCOL3A1K.

FIG. 5: Oligo sequences for cloning of CO1A1 (C1A1—FW—SEQ ID NO:39;C1A1—RV1—SEQ ID NO:40; C1A1—RV2—SEQ ID NO:41; C1A1—RV3—SEQ ID NO:42;N—X-FW—SEQ ID NO:43; N—X-RV—SEQ ID NO:45). The bottom sequence is theadaptor after annealing the top strand (SEQ ID NO:45) with the bottomstrand (SEQ ID NO:46).

FIG. 6: Cloning strategy

FIG. 7: vector pCOL1A1-1

FIG. 8: The expected COL1A1-1 sequence (SEQ ID NO:47). The singlyunderlined sequences correspond to the N terminal sequences obtained forCOL1A1 fragments. The double underlined sequences share this sequence.Both fragment are extended at the N terminus by EA.

FIG. 9: vector pCOL1A1-2

FIG. 10: Expected COL1A1-2 sequence (SEQ ID NO:48)

FIG. 11 vector pCOL1A1-3

FIG. 12 Expected COL1A1-3* sequence (SEQ ID NO:49)

FIG. 13: MGPR sequence in the expected COL1A1-2 sequence (SEQ ID NO:50).The singly underlined sequences correspond to the N terminal sequencesobtained for COL1A1-1 fragments. The double underlined sequences is theMPPR sequence.

FIG. 14: SDS poly acryl amide gel electrophoresis of GATO4. The gel wasstained with Coommassie Brilliant Blue. In the left most lane themolecular marker protein mix is visible. From top to bottom the bandscorrespond to molecular weights of 94, 67, 43, 30, 20 and 14.4 kD. Thesecond and third lane from the left show GATO4 after purification.

FIG. 15: SDS poly acryl amide gel electrophoresis of GATO5. The gel wasstained with Coommassie Brilliant Blue. In the right most lane themolecular marker protein mix is visible. From top to bottom the bandscorrespond to molecular weights of 94, 67, 43, 30, 20 and 1404 kD. Thesecond and third lane from the right show GATO5 after purification.

FIG. 16 SDS poly acryl amide gel electrophoresis of expression productcol1A1-2. The gel was stained with Coommassie Brilliant Blue. In theleft most lane the molecular marker protein mix is visible. From top tobottom the bands correspond to molecular weights of 94, 67, 43, 30, 20,1 and 14.4 kD.

FIG. 17 SDS poly acryl amide gel electrophoresis of expression productcol1A1-1 in which MGPR sequences have been mutated to RGPM. The gel wasstained with Coommassie Brilliant Blue. In the left most lane themolecular marker protein mix is visible. From top to bottom the bandscorrespond to molecular weights of 94, 67, 43, 30, 20, 1 and 14.4 kD.

FIG. 18 SDS poly acryl amide gel electrophoresis of expression productcol1A1-2 in which MGPR sequences have been mutated to RGPM. The gel wasstained with Coommassie Brilliant Blue. In the left most lane themolecular marker protein mix is visible. From top to bottom the bandscorrespond to molecular weights of 94, 67, 43, 30, 20, 1 and 14.4 kD.

REFERENCES CITED IN EXAMPLE 1

-   [1] Capello, J. & Ferrari, F. (1994) in: Plastics from microbes    (Mobley, D. P., ed.) Hanser, Munich, pp. 35-92-   [2] Strausberg, R. L. & Link, R. P. (1990) TIBTECH 8: 53-57.-   [3] Maniatis T., Fritsch, E. F. & Sambrook, J. (1982) Molecular    cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold    Spring Harbor, N.Y.-   [4] Manual of the Pichia Expression Kit Version E (Invitrogen, San    Diego, Calif., USA).-   [5] Glumoff, V., Mäkelä J. K. & Vuorio, E (1994) Cloning of cDNA for    rat proα1(III) collagen mRNA. Increased expression of type III    collagen gene during induction of experimental granulation tissue.    Biochim Biophys Acta 1217: 41-48.    -   EMBL/GenBank accession number X70369.-   [6] Sanger, F., Nicklen, S. & Coulson. A. R. (1977) Proc. Natl.    Acad. Sci. USA 74: 5463-5467.-   [7] Becker, D. M. & Guarente, L. (1991) High efficiency    transformation of yeast by electoporation. Methods in Enzymology,    vol. 194: 182-187.-   [8] Lee, F. J. S. (1992) Biotechniques 12 (5): 677-   [9] Laemmli, U. K. (1970) Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature 227: 680-685.-   [10] Schmitt, M. E., Brown, T. A. and Trumpower. B. L. (1990) A    rapid and simple method for preparation of RNA from Saccharomyces    cerevisiae. Nucleic Acids Res. 18 (10): 3091.-   [11] Scorer, C. A., Clare, J. J., McCombie, W. R., Romanos, M. A. &    Sreekrishna, K. (1994) Rapid Selection using G418 of high copy    number transformants of Pichia pastoris for high-level foreign gene    expression. Bio/Technology 12: 181-184.-   [12] Butkowsky R. J., Noelken. M. E. & Hudson, B. G. (1982)    Estimation of the size of colagenous proteins by electrophoresis and    gel chromatography. Meth. Enzymol. 82: 410-423.-   [13] De Wolf, F. A. & Keller R. C. A (1996) Characterization of the    helical structures in gelatin networks and model polypeptides by    circular dichroism. Progr. Colloid Polym. Sci. 102: 9-14

Example 2 Control

Preparation of Silver Bromide Crystals with Conventional Regular TypeGelatin.

-   -   Nucleation: At a temperature of 35° C. in a reaction vessel        containing 2.1 g/l regular gelatin (a standard deionised LAG        bone gelatin from PB Gelatins, Tessenderlo, Belgium) and 7.3 mM        potassium bromide the pH is adjusted to a value of 5.5 by sodium        hydroxide or sulphuric acid. By single jet addition an aqueous        solution of 144 mM silver nitrate is added at a constant rate in        a period of 10 sec while vigorously stir-ring. After addition of        the silver nitrate the gelatin concentration in the reaction        mixture has become 2.0 g/l and the bromide concentration 1 mM.    -   Ripening: After nucleation the content of the vessel is        transferred to a ripening vessel, where the temperature is        increased gradually to a value of 75 C and the bromide        concentration is increased to 15 mmol, by adding a 3.4 M        potassium bromide solution. The ripening is continued for 56        minutes after which a standard deionised gelatin is added up to        a concentration of 5 g/l: after 58 minutes the Ostwald ripening        is strongly reduced by adding a solution of methyl phenyl        tetrazole and cooling to room temperature. A sample of the        prepared emulsion was analyzed by direct transmission electron        microscopy as well as by replica thereof.    -   Result: As can be seen in table II very low % of tabular grains        is formed.

Example 3 Control

Preparation of Silver Bromide Crystals with Conventional HydrolysedGelatin.

-   -   Nucleation: The nucleation is performed applying the same        conditions (also pH=5-0.5) as in example 2 except that the        gelatin in the reaction mixture is replaced by a conventional        hydrolysed gelatin sample (also deionised and supplied by Nitta        Gelatin in Japan).    -   Ripening: The ripening is done according the same procedure as        is used in example 2.    -   Result: A medium % tabular grains of ca 40% is shown in table        II.

Example 4 Control

Preparation of Silver Bromide Crystals with Oxidized Gelatin.

-   -   Nucleation: The nucleation is performed applying the same        conditions (also pH=5-0.5) as in example 1 except that the        gelatin in the reaction mixture is replaced by a conventional        oxidized gelatin sample (supplied by PB Gelatins Tessenderlo in        Beigium).    -   Ripening: The ripening is done according the same procedure as        is used in example 2.    -   Result: A high % tabular grains of 70% with an average aspect        ratio of 5:1 is shown in table II for the oxidized gelatins at        pH 5.5. The better result than with the hydrolysed and regular        gelatin is to be explained due to the lower methionine content        of this gelatin (11 μmol/gram gelatin vs. 50-60 μmol/gram for        the conventional gelatins).

Example 5 This Invention

Preparation of Silver Bromide Crystals with Invented Native RecombinantGelatins.

-   -   Nucleation: The nucleation is performed applying the same        conditions (also pH=5-0.5) as in example 2 except that the        gelatin in the reaction mixture is replaced by the invented        native COL3A1 gelatin sample.    -   Ripening: The ripening is done according to the same procedure        as is used in example 2.    -   Result: A high % tabular grains of more than 85% with an average        aspect ratio of 5:1 is shown in table II.

Example 6 Control

Preparation of Silver Bromide Crystals with Conventional Regular Gelatinat Different pH (Standard Deionised IAG bone Gelatin from PB Gelatins.Tessenderlo in Belgium.

-   -   Nucleation: the nucleation is performed applying a pH=7        condition while the other conditions remained the same as in        example 2.    -   Ripening: The ripening is done according to the same procedure        as is used in example 2 except the pH remained the same i.e.        pH=7 as during the nucleation.    -   Result: No tabular grains of aspect ratio larger than 5 resulted        as is shown in table II for conventional commercial gelatin.

Example 7 Control

Preparation of Silver Bromide Crystals with Conventional HydrolysedGelatins at a Different pH (Nitta Gelatins in Japan).

-   -   Nucleation: The nucleation is performed applying a different        pH=7 condition while the other conditions remained the same as        in example 2.    -   Ripening: the ripening is done according to the same procedure        as is used in example 2 except the pH remained the same at pH=7        as during the nucleation.    -   Result: A very low % tabular rains around 5% resulted as is        shown in table II.

Example 8 This Invention

Preparation of Silver Bromide Crystals with Invented Native RecombinantGelatin at a Different pH.

-   -   Nucleation: The nucleation is performed applying a different pH        condition i.e. pH=7 while the other conditions remained the same        as in example 4.    -   Ripening: The ripening is done according to the same procedure        as is used in example 4 except the pH remained the same i.e. at        pH=7 as during the nucleation.    -   Result: A very high % tabular grains ca 80% is surprisingly        found at this condition which is clearly higher than the        state-of-the-art gelatins as is shown in table II.

Example 9 Relation Between Binding Strength and Tabular Grain Morphology

45 mg gelatin is accurately weighed and 15 g 0.1M phosphate bufferpH=7.00 containing 0.1M potassium nitrate, is added. The solution isplaced in a waterbath at 45° C. for 15 minutes. The solution is cooledto room temperature (23° C.). 10 ml of this pH 7.0 phosphate buffersolution (containing gelatin) is mixed at 23° C. with 100 μl 10.5 mMsilver nitrate. The potential of this solution entitled as “vAg” ismeasured using an Ag electrode (Orion model 97-81) against an Ag/AgClreference double junction electrode (Orion model 90-02). The same buffersolution without gelatin is also mixed with the silver nitrate solutionand the potential “vAg” measured by the same method. The differencebetween the two measured potentials is calculated and expressed as“delta vAg” being the binding affinity of gelatin for the Ag-ion. TableII below contains the tested peptizers, the % tabular grains and thegelatin binding affinities “delta VAg” for pH 5.5 and pH 7 in which, thecriteria for tabularity has been defined at aspect ratio >5:

% tabular % tabular at at Bind. strength nucleation/ nucleation/ Type“delta vAg” ripening ripening gelatin (mV) pH = 5.5 pH = 7.0 oxidized55.4 73 —* hydrolysed 74.4 42 5 regular 78.5 1 0 nat. rec COL3 69.5 3779 *not measured

1-3. (canceled)
 4. A process for producing a collagen-like polypeptide,said polypeptide comprising [Gly-X-Y]n repeats, wherein Gly stands forglycine, X and Y represent any amino acid and n is an integer andselected such that the length of the polypeptide is at least 2.5 kDa andwherein the amino acid sequence of said polypeptide comprises more than4 different amino acids and wherein said purified polypeptide is free ofhelix structure. said process comprising the following steps: (a)expressing a nucleic acid sequence encoding said polypeptide in afungus, (b) growing said fungus in culture medium, (c) purifying saidpolypeptide from said culture medium.
 5. The process according to claim4 wherein said fungus is a methylotrophic yeast.
 6. The processaccording to claim 4 wherein said nucleic acid sequence encoding saidpolypeptide is free of procollagen and telopeptide encoding sequences 7.The process according to claim 4 wherein at least 0.95 gram/liter ofsaid polypeptide are produced.
 8. The process according to claim 4wherein at least 3 gram/liter of said polypeptide are produced.
 9. Theprocess according to claim 4 wherein the said polypeptide is secreted.10. The process according to claim 4 wherein the fungus is free ofactive post translational processing mechanism for processing collagenlike polypeptide to fibrils.
 11. The process according to claim 4wherein the fungus is free of active post translational processingmechanism for processing collagen like polypeptide to triple helices.12. The process according to claim 5 wherein the methylotrophic yeast isselected from Hansenula and Pichia.
 13. The process according to claim12 wherein the methylotrophic yeast is Pichia pastoris.
 14. The processaccording to claim 4 wherein the expression product is isolated andpurified until it is substantially free of other protein, polysaccharideand nucleic acid.
 15. A purified polypeptide obtained by the methodaccording to claim 4, said polypeptide comprising or consisting of anamino acid sequence that has at least 90% sequence identity with anamino acid sequence selected from the group consisting of SEQ ID NO:33(=P), SEQ ID NO: 34 (=P4) SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 48and SEQ ID NO:
 49. 16. The purified polypeptide according to claim 15,said polypeptide comprising or consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 33(=P), SEQ ID NO: 34(=P4) SEQ ID NO: 38, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO:
 49. 17.The purified polypeptide according to claim 15, said polypeptidecomprising less than 100 ppm nucleic acids.
 18. The purified polypeptideaccording to claim 15, said polypeptide comprising less than 5%polysaccharides.